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The stage analysis and countermeasures of coal spontaneous combustion based on “five stages” division The "three stages" division of coal spontaneous combustion is fuzzy and lacks adequate risk and warning levels corresponding to its divisions; additionally, the targeted prevention measures for each stage have not been described. To address the shortcomings of the "three stages" division, the "five stages" division was proposed to more clearly analyze the stage changes of the spontaneous combustion of coal. The "five stages" method divides the process of the spontaneous combustion of coal into five stages, including: the latent stage, heat accumulating stage, evaporation stage, active stage, and hypoxic stage. The critical point of each stage was determined using adiabatic oxidation experiments and programmed heat experiments. As the critical point of the latent stage, the temperature of zero activation energy is approximately 55-70˚C. In the heat accumulating stage, the critical point is the temperature (approximately 90˚C) where the external moisture of coal evaporates violently while the internal moisture of coal has not yet fully evaporated. During the evaporation stage, the temperature (approximately 105˚C) where the internal moisture has evaporated completely represents the end of this stage and the start of the active stage (105-170˚C). When the oxygen concentration drops to 5%, the spontaneous combustion of coal enters the hypoxic stage. Thus, an oxygen concentration of 5% represents the critical point of the start of the hypoxic stage (above 170˚C). After the analysis of each stage, risk and warning levels were determined. Considering the major prevention measures of the spontaneous combustion of coal, a staged warning and disposal table was created.OPEN ACCESSCitation: Zhu H, Sheng K, Zhang Y, Fang S, Wu Y (2018) The stage analysis and countermeasures of coal spontaneous combustion based on "five stages" division. PLoS ONE 13(8): e0202724. # Introduction The mechanism of the spontaneous combustion of coal has been studied since the seventeenth century and, to date, a number of coal spontaneous combustion theories have been proposed. These include: the pyrite-related cause theory, the bacteria-related cause theory, the phenolicgroups-related cause theory and the coal-oxygen-compound theory. Among them, the coal-oxygen-compound theory has been widely accepted by scholars because the adsorption of oxygen to coal and the production of an exotherm, which is core of this theory, has been proven by experiments. Some scholars [bib_ref] Coal fire mapping from satellite thermal IR data-a case example in Jharia..., Chatterjee [/bib_ref] [bib_ref] Experimental studies of spontaneous combustion and anaerobic cooling of coal, Deng [/bib_ref] [bib_ref] A review on numerical solutions to self-heating of coal stockpile: mechanism, theoretical..., Zhang [/bib_ref] have supported this theory and described and discussed the mechanisms of the spontaneous combustion of coal as follows: (1) the percolation of air through coal results in a measurable rise in temperature, which is caused by a series of adsorptive, absorptive, and chemical processes; (2) the heat generated gives rise to an increase in the temperature of the coal, which accelerates the rate of coal oxidation; (3) these actions cause the coal to self-heat, and if conditions are favorable, spontaneous combustion will occur. In previous studies of coal-oxygen compound reactions, various parameters of coal spontaneous combustion processes were studied using adiabatic oxidation tests, programmed heat experiments and gas chromatography [bib_ref] Experimental studies of spontaneous combustion and anaerobic cooling of coal, Deng [/bib_ref] [bib_ref] A review on numerical solutions to self-heating of coal stockpile: mechanism, theoretical..., Zhang [/bib_ref] [bib_ref] Test method of critical temperature of coal spontaneous combustion based on the..., Zhong [/bib_ref] [bib_ref] Calculation of ignition times under adiabatic conditions by activation energy, Li [/bib_ref] [bib_ref] Low-temperature reactivity of coals for evaluation of spontaneous combustion propensity, Kim [/bib_ref] [bib_ref] Study on Adiabatic Oxidation Characters of Coal with Applying a Constant. Temperature..., Lu [/bib_ref] [bib_ref] Laboratory Study on the Rising Temperature of Spontaneous Combustion in Coal Stockpiles..., Lu [/bib_ref]. Based on the coal-oxygen-compound theory, many scholars believe that the phenomenon of coal spontaneous combustion may generally be divided into three stages: the incubation stage, the self-heating stage and the combustion stage. Each stage is separated by variations in temperature and concentrations of the main index gases (CO 2 , CH 4 , H 2 O, CO). Cole [bib_ref] Transformations of iron minerals during coal oxidation, Cole [/bib_ref] discussed a detailed reaction mechanism while researching the influence of pyrite on the spontaneous combustion of coal and noted that mineral transformations were closely related to the generation of free radicals in coal. Based on the "three stages" division, Quobtained the stage characteristics and critical temperature points of low-and high temperature-stages in the process of the spontaneous combustion of coal using a simultaneous thermal analysis experiment, Fourier transform infrared spectrum analysis and a programmed heat experiment. Xuinvestigated the heat release characteristics, changes in functional groups and oxygen consumption in the process of coal spontaneous combustion through adiabatic oxidation and Fourier transform infrared spectrometry analysis, and they found that the heat release of coal has obvious segmentation characteristics. Most previous studies have been based on the "three stages" division and mainly concentrated on the variation laws of characterization parameters, such as oxygen consumption, activation energy, and functional groups, but they have not proposed corresponding disposal measures and warnings for all stages during the process of the spontaneous combustion of coal. The "three stages" division of coal spontaneous combustion is based on the coal-oxygen compound theory, which is fuzzy and contains no explicit division points. The determination of the spontaneous combustion stage often requires testing the concentrations of index gases or assessing temperature changes. Meanwhile, coal with different coal qualities (i.e., different metamorphic grades) have different critical temperatures of spontaneous combustion and a large range of critical temperature values. More practical and universal stage divisions should involve variations in coal characteristics. Thus, here, the authors propose a "five stages" division and determine the limits for each proposed stage. As a practical result, stage warnings and disposal mechanisms are established, which may help the fire prevention departments of coal mines take effective and corresponding preventive measures before a particular stage of coal combustion. # Methods ## Adiabatic oxidation experiment We used an adiabatic oxidation experiment to obtain the temperature of zero activation energy. This experiment uses a small coal adiabatic oxidation test system, which was created at the Safety Engineering Faculty, China University of Mining & Technology (Beijing) and is shown in [fig_ref] Fig 1: The small coal adiabatic oxidation testing system [/fig_ref] The entire experiment system consists of a gas source system, gas path, preheat gas system, coal sample tank, adiabatic oven, temperature monitoring system, and data acquisition system. ## Coal samples. To assess the variable characteristics of the coal, we collected three types of coal samples from the Renlou Coal Mining Co., Ltd (NO. [bib_ref] Coal fire mapping from satellite thermal IR data-a case example in Jharia..., Chatterjee [/bib_ref]. The proximate analysis of the coal samples were tested, and the results are shown in [fig_ref] Table 1: The industry index values of three coal samples [/fig_ref]. The results indicate that sample NO.1 is a long flame coal, sample NO.2 is a gas coal, and sample NO.3 is a charred coal. Experimental procedures. Prior to the experiment, 200 g of a pulverized coal sample were brought into the tank and dried for 16 hours in a nitrogen-rich atmosphere (with a nitrogen flow rate of 120 mL/min), and the adiabatic oven was set at 105˚C. After drying, once the temperature of the coal sample had dropped down to room temperature, the coal sample was transferred into the adiabatic oxidation tank. Then, the temperature program was switched to automatic tracking mode, the nitrogen valve was closed, and the system started pumping in oxygen at a flow rate of 60 mL/min. The computer recorded and saved the changes in coal temperature data over time. ## Isothermal heating experiment Original coal samples were collected from the Renlou Coal Mining Co., Ltd.; their original moisture content was 2.15%. These samples were then used to make coal samples with water contents of 5.6, 7.2, 9.35, 11.8, 13.1 and 15.25%, and 200 g of each sample were used for the experiment. The experimental device used here is the small coal adiabatic oxidation test system. Including the original coal sample, a total of seven coal samples with different moisture contents were placed into the coal sample tanks; then, the tanks were placed into an adiabatic oven and underwent isothermal heating at 150˚C (The oven temperature can be held constant from 40 to 200˚C, within an accuracy of ±0.5˚C. In each experiment, the oven is first turned on and preset at a certain temperature. After about 30 min, the isothermal condition is reached in the oven.). To avoid the influence of the heat released from the coal oxidation of the coal sample itself, a 120 mL/min flow of nitrogen was inlet into the tanks. A computer recorded the temperature and heating time of each coal sample. ## Programmed heat experiment Coal samples. To assess the different coal qualities, six coal samples (A/B/C/D/E/F) collected from six different places in the Renlou Coal Mining Co., Ltd. were used in the programmed heat experiment. The proximate analysis of the coal samples were tested, and the results are shown in [fig_ref] Table 2: The proximate analysis test results [/fig_ref]. These data suggest that the coal qualities of the six coal samples differ. Experimental procedures. The coal samples were crushed under the protection of nitrogen, and a total of 200 g (in 40-g aliquots) were sieved to particle sizes of 1.23-1.6, 1.6-2.5, 2.5-3.5, 3.5-5, and 5-7 mm. After obtaining uniform mixtures, the coal samples were placed into a tank in a nitrogen-rich atmosphere (with a nitrogen flow rate of 120 mL/min). The tanks containing coal samples were placed in an adiabatic oven to dry for 15 h at 105˚C. After the completion of the drying procedure, the program turned off the nitrogen and opened an air valve (at the same rate as above) for the programmed heat experiment. The compositions of gaseous products were analyzed at each 10˚C step by gas chromatography. The temperature rising program was set as follows: the temperature first remained at 25˚C for 30 min, then increased to 35˚C in 1 min, and finally rose to 185˚C at a rate of 0.5˚C/min. When the temperature reached 200˚C, the experiment ended. ## "five stages" of coal spontaneous combustion ## The latent stage Here, the time interval from the exposure of the coal seam to the air to the beginning of the increase in coal temperature is called the latent stage. This stage includes two substages, and the first one is the traditional latent stage. The main reaction between coal and oxygen is physical adsorption, which releases a small amount of heat and adsorbs oxygen to form unstable oxides or oxygen-containing free radicals. The second substage is the preparation stage, which leads up to a critical point of the heat accumulating stage. In the preparation stage, the chemical adsorption increases and heat production is greater than heat dissipation; at the same time, the coal temperature gradually increases at a lower rate. In this stage, CO and CH 4 usually appear and slowly increase, CO 2 slowly increases, and oxygen essentially remains stable. According to the coal-oxygen-compound theory, coal is a porous medium, and the strong adsorption of air may occur within it. The original heat production is mainly due to the physical and chemical adsorption of oxygen on coal at the beginning of the spontaneous combustion of coal. The basic dynamic equation in the process of the spontaneous combustion of coal can be described as follows [bib_ref] Self-activation theory of spontaneous combustion of coal, Li [/bib_ref] : [formula] cr @T @t ¼ QrAexp À E RT þ lr 2 T À cr oxygen v @T @x À H w @C w @tð1Þ [/formula] In the above equation, c is the specific heat capacity of the sample material, J kg -1 K -1 ; ρ is the density of the sample, kg m -3 ; T is the sample temperature, K; t represents time, s; Q is the heat of oxidation per unit mass under standard state conditions, kJ kg -1 ; A refers to the former factor, s -1 ; E is the activation energy, kJ mol -1 ; R is the ideal gas constant, 8.314 J K -1 mol -1 ; ν is the flow rate of oxygen, m s -1 ; H w is either dry or humid heat, J m -3 s -1 ; λ is the coefficient of heat conduction, W m -1 K -1 ; and C w is the moisture content of coal, %. There are four terms on the right side of Eq (1); the 2 nd , 3 rd , and 4 th terms represent heat conduction, heat convection and transfer and water evaporation, respectively. These are all external factors of the spontaneous combustion of coal. The first term is the kinetic expression of the heat produced by the oxidation of coal at low temperatures, which is the inner driving power of coal spontaneous combustion. In the latent stage, coal is at a low temperature similar to the surrounding temperature; thus, the 2-4 th terms can be ignored. Eq (1) can be simplified as Eq (2) [bib_ref] Tendency of spontaneous combustion of coal based on activation energy, Lu [/bib_ref] : [formula] cr @T @t ¼ QrAexp À E RTð2Þ [/formula] Eq (3) can be obtained by the logarithm transformation and simplification of Eq (2) [bib_ref] Tendency of spontaneous combustion of coal based on activation energy, Lu [/bib_ref] : [formula] ln @T @t ¼ À E RT þ ln QA cð3Þ [/formula] The activation energy E is the core parameter of coal low-temperature oxidation power, on behalf of the low-temperature oxidation capacity, namely, the coal spontaneous combustion tendency. Therefore, the change in activation energy should be regarded as the critical index of the latent stage. Lutested the range of different coal samples using an adiabatic oxidation experiment and obtained a relationship between activation energy and temperature. In the thermal range of 55-70˚C, the activation energy of the coal sample changes from positive to negative [bib_ref] Effect of moisture content on the R70 self-heating rate of Callide coal, Beamish [/bib_ref]. This means that the activation energy exists at a zero value in the given range. Different coal samples reach zero activation energy at different temperatures. The zero activation energy theory explains the process by which the coal gains heat from physical and chemical adsorption until it can produce heat itself. Therefore, the zero activation energy [bib_ref] Kinetic parameters of oxidation of bituminous coals from heatrelease rate measurements, Jones [/bib_ref] can be calculated to determine the critical point between the latent stage and the heat accumulating stage. We also used adiabatic oxidation experiments to obtain the temperature of zero activation energy. Based on our experiment, we calculated the activation energy of the coal samples when the temperature increased at each 10˚C step to obtain the temperature-dependent activation energy curve, as shown in ## The heat accumulating stage The period from the active oxidation heat release to the point at which water evaporates violently is the second stage of the spontaneous combustion of coal. Here, this is called the heat accumulating stage. At this stage, the heat release from chemical adsorption still plays a considerable role, but the oxidation heat release rates of coal and oxygen increase rapidly, and the CO and CO 2 emissions continue to increase. During this stage, the external water of coal evaporates faster with the increasing temperature, while the internal water does not evaporate yet. The steam generated by the evaporation of external water will remove some heat, but it may be partially absorbed by coal pores that will retain the heat and fill the coal pores, thus producing a heat-preserving effect similar to the "greenhouse effect". Beamish [bib_ref] Effect of moisture content on the R70 self-heating rate of Callide coal, Beamish [/bib_ref] found that a coal sample with a higher moisture content needs more time to cool down from 110˚C to 40˚C. This demonstrates the existence of a heat preservation effect in coal related to moisture evaporation. During the early stage of the spontaneous combustion of coal, external moisture begins to evaporate (at approximately 50˚C) with a catalytic action [bib_ref] Coal oxidation at low temperatures: oxygen consumption, oxidation products, reaction mechanism and..., Wang [/bib_ref] [bib_ref] Modelling of spontaneous combustion of coal with moisture content included, Arisoy [/bib_ref] [bib_ref] Effects of moisture in coal on pulverized coal combustion characteristics, Kurose [/bib_ref] [bib_ref] The role of moisture in the self-heating of low-rank coals, Clemens [/bib_ref] [bib_ref] Steam-drying of coal. Part 1. Modeling the behavior of a single particle, Chen [/bib_ref] [bib_ref] Spontaneous combustion of carbonaceous stockpiles. Part II. Factors affecting the rate of..., Smith [/bib_ref] , because moisture is involved in the formation of free radicals and plays an important role in promoting the formation of peroxide complexes [bib_ref] Effects of moisture on spontaneous combustion of coal, Liang [/bib_ref]. Water evaporation in coal can also lead to the decomposition of peroxide complexes that will accelerate combustion. Meanwhile, due to the dynamic action of water vapor, many pores or fissures will be formed within coal, so that the surface area of coal and the contact area between coal and oxygen will increase. At approximately 40-50% of the moisture holding capacity of the coal, above this critical level of moisture content, the heat produced by oxidation is dissipated by moisture evaporation and coal self-heating is significantly delayed [bib_ref] Effect of moisture content on the R70 self-heating rate of Callide coal, Beamish [/bib_ref]. When the moisture content is higher than a critical value, a layer of a water-containing liquid membrane will form at the coal surface. This membrane may hinder the contact of coal and oxygen and prevent their interaction. This situation is, however, rare. Normally, the moisture content in coal is lower than the critical value. To find the critical temperature point of the heat accumulating stage, we must determine the starting point at which the moisture content has an important impact on the heating process of coal. Therefore, we designed an isothermal heating experiment for coal samples with There is a huge internal surface area in the coal. After water evaporation from the coal, more pore channels or fissures are formed, so that the coal has a larger internal surface area, which is beneficial for more oxygen to enter the coal for internal oxidation. The predominance of the endothermic reaction in the primary stage of coal self-heating is mainly due to the evaporation of moisture in the coal. After the water in the coal evaporates, the oxidation activation center of the coal surface is increased. The second phase after the evaporation of water, that is, after the dehydration phase, the reaction becomes more intense as more points of evaporation water are distributed. According to the study of oxidation under adiabatic conditions (simulating the conditions of the mine), the evaporation of moisture can make the coal oxidation more intense. Initially, the oxidation was carried out in large pores. After the water evaporates, oxidation takes place in the medium and micropores, which have a large surface area, which greatly increases the specific surface area of the reaction and accelerates oxidation. As seen in [fig_ref] Fig 3: Thermal curves of temperatures of coal samples with different moisture contents [/fig_ref] the temperatures of coal samples increase rapidly up to 90˚C, although the rates of different samples are slightly different. The heating rate of the coal samples becomes slower when the temperature crosses 90˚C, and it even stagnates when it reaches 100˚C. This phenomenon demonstrates that the moisture contained in coal begins to have a serious impact on the heating process of coal samples at approximately 90˚C. The reason for this is that the external moisture of coal samples begins to evaporate intensely at 90˚C. Thus, the temperature at which the evaporation of external moisture occurs represents the critical point between the heat accumulating stage and the following evaporation stage. [fig_ref] Fig 3: Thermal curves of temperatures of coal samples with different moisture contents [/fig_ref] indicates that the heating rate of the coal samples with a moisture content of 5.6% and 7.2% are the fastest and are greater than that of the original coal sample; thus, the optimum moisture content of a coal sample falls in the range of 2.15-7.2%. ## The evaporation stage During this stage, due to the heat accumulation in the previous stage, the temperature is increasing, the content of active moleculeswithin the coal has largely increased, their collisions are more frequent, and the reaction rate between coal and oxygen is higher [bib_ref] Laboratory study on the spontaneous combustion propensity of lignite undergone heating treatment..., Tang [/bib_ref]. At approximately 90-105˚C, the external water has almost completely evaporated, and the internal water also starts to evaporate. Eqindicates that the heat generation caused by the process of the spontaneous combustion of coal also includes the humid heat of water. However, water is often absent in a coal sample before the adiabatic oxidation experiment because the coal sample has been dried beforehand; thus, the moisture evaporation stage is often ignored in the analysis and research of the adiabatic oxidation of coal. Because this stage appears in a narrow temperature range and has no obvious characteristics, it is easy to ignore. However, the evaporation stage plays an important role in delaying the further active stages of the spontaneous combustion of coal. As shown in [fig_ref] Fig 3: Thermal curves of temperatures of coal samples with different moisture contents [/fig_ref] the thermal range at which the heating rate is almost stationary is exactly 90-105˚C. The greater the moisture content is, the longer duration of this phase is (i.e., the heating rate is almost stationary). The coal samples with moisture contents of 13.1% and 15.25% show much longer durations of this stage than the other samples. At 105˚C, the internal moisture has almost completely evaporated. The temperature stagnation at this stage is due to the fact that the internal moisture captures some of the heat generated by the oxidation of coal, and the heat requirement of the evaporation of internal moisture is much greater than that of external moisture. Therefore, the effect of the evaporation of internal moisture can delay the active stage, thus providing time for workers to prepare prevention measures. ## The active stage This stage is ascribed to the thermal range of 105-170˚C. Due to the complete desorption of both the internal and external moisture in coal during the foregoing stages, the reaction between coal and oxygen begins, and oxygen consumption, heat release and gas production show qualitative changes. Many researchers have proposed that the coal-oxygen recombination reaction occurs in the temperature range of 105-170˚C and that lower degrees of coal metamorphism cause faster temperature growth [bib_ref] Prediction model of coal spontaneous combustion critical point and the characteristics of..., Tan [/bib_ref] [bib_ref] Characteristics and model of loose coal low-temperature oxygen consumption rate by multiple..., Zhu [/bib_ref] [bib_ref] The relationship between oxidation kinetics characteristic parameters of coal adiabatic progress and..., Zhu [/bib_ref]. At this stage, the changes in the concentrations of O 2 , CO, C 2 H 4 and other gases are clearly related to the intensity of the reaction between coal and oxygen. Therefore, a programmed heat experiment for coal samples with different coal qualities was designed to verify the existence of the active stage. The temperaturedependent variations in the contents of CO and C 2 H 4 are shown in Figs 4 and 5, respectively. As shown in the above figures, the CO contents in all samples increase rapidly starting at approximately 110˚C, while the C 2 H 4 contents in all samples increase rapidly starting at approximately 115˚C. During the spontaneous combustion of coal with different degrees of coalification, there are differences in the consumption rates of O 2 and the production rates of CO, CO 2 and C 2 H 4 . Although the critical temperature, i.e., the temperature at which the rate drastically changes, varies between CO and C 2 H 4 , the critical temperatures of different samples still fall within the range of 100-115˚C. This temperature range happens to represent the evaporation temperature at which both the external and internal moisture evaporate completely. Thus, the above analysis demonstrates that when both the external and internal moisture evaporate completely, the reaction between coal and oxygen enters a very active stage because the heat generated by the reaction is no longer used to evaporate moisture. ## The hypoxic stage During this stage, two methods of coal combustion may occur. The first occurs when the O 2 supply is constant, and the second occurs when it increases. The temperatures of the coal samples increase rapidly during the period from the active stage to coal ignition. If the oxygen supply is sufficient, a flame will appear, and the coal sample will start to burn. At this time, a large amount of high-temperature smoke is generated. This smoke contains CO, CO 2 and hydrocarbons, among other substances [bib_ref] Combustion metamorphic events resulting from natural coal fires, Sokol [/bib_ref] [bib_ref] Combustion metamorphism in the Nabi Musa dome: new implications for a mud..., Sokol [/bib_ref]. However, if the oxygen supply is insufficient, the coal sample will develop smoldering smoke without a flame. In general, as the activated molecules in the coal seam increase with increasing temperature, more oxygen will be required. To maintain the combustion of coal, the supply of oxygen must be increased. However, during the experimental process, the oxygen supply is constant because the oxygen flow is stable. In addition, during the practice of coal mining, the air quantity and air pressure are constant, which means that the air velocity is constant in galleries and that air leakage or flow tends to be constant. Therefore, the oxygen supply for the spontaneous combustion of coal is not expected to increase during the practice of coal mine production. If so, the spontaneous combustion of coal will enter the hypoxic stage. In general, when the oxygen content is less than 5%, the coal in the fire area remains in a smoldering state and flame combustion cannot exist [bib_ref] Volume fraction gradient criterion of oxygen in a smoldering state of coal..., Wang [/bib_ref] ; thus, an oxygen concentration of 5% can be used as a threshold of the spontaneous combustion of coal entering the hypoxic stage. Based on the programmed heating data, the oxygen concentration curves of coal samples A-F are drawn in [fig_ref] Fig 6: The relationship between oxygen concentration and temperature [/fig_ref] As seen in this figure, when the oxygen content is approximately 5%, the temperatures of all samples are above 170˚C. Thus, the critical temperatures of the hypoxic stage of the six samples are all beyond 170˚C. However, the spontaneous combustion characteristics of coal are greatly affected by coal quality, thus affecting the lower thermal limit of the hypoxic stage. The six coal samples assessed in this paper cannot represent all types of coal; thus, the critical temperatures of specific coal samples should be identified using programmed heat experiments, and the effects of coal quality rank on the critical temperature of the hypoxic stage require further study. ## Establishment of staged disposal measures for the spontaneous combustion of coal ## Analysis of various self-ignition prevention applications The prevention of coal self-ignition mainly include three types of procedures: pressure equalizing, anti-fire material injection and sealing a fire zone. Pressure equalizing. This fire prevention theory suggests equalizing the pressure of a leakage path between an intake and outlet air ventilation stream, with the purpose of either eliminating or reducing the air leakage. The essence of pressure equalizing is to set air pressure regulators or adjust the ventilation system to reduce the pressure between the both ends of the leakage path, thereby reducing access to the coal accumulation area. As a result, one can inhibit or eliminate the coal spontaneous combustion phenomenon. The pressure equalizing technology has the advantages of simple principles and negates the need to ascertain the specific locations of fire sources. It is possible to extinguish a large coal fire using pressure equalizing. The mentioned technology is just an air regulation method that poses no hazard to the mine staff, which requires little expense and has no effect on normal production. In addition, compared with the injection of anti-fire material, pressure equalizing is only related to air, so it is not restricted by water, soil and provenance, and it can continuously prevent fires for a long time in a coal mine. It is applicable to the heat accumulation stage and evaporation stage during the early coal spontaneous combustion process because the scope of the fire is smaller in this period, and fewer harmful gases are produced. "Five stages" division of coal spontaneous combustion and its countermeasures Anti-fire material injection. Anti-fire material injection refers to injecting an inert gas, gel material, three-phase foam or other material into the fire area [bib_ref] New approaches for increasing the incubation period of spontaneous combustion of coal..., Tripathi [/bib_ref] [bib_ref] A new approach to control a serious mine fire with using liquid..., Zhou [/bib_ref] [bib_ref] Research and development of foamed gel for controlling the spontaneous combustion of..., Wang [/bib_ref]. The main purposes of this method are to dilute the oxygen concentration, reduce the temperature and isolate the contact surface between coal and oxygen. Inert gas injection: The advantages of inert gas injection are that it can cause methane and other flammable gases to lose their explosiveness by reducing the oxygen concentration and that it has no corrosive effect on mining devices or negative effects on the health of mine workers. Its disadvantages include easy gas diffusion, which can be followed by possible air leakage or inert gases spreading randomly, the need for the nitrogen injection machine to be regularly maintained, and its poor cooling extinguishing effect on the fire area. Gel material injection: The advantages of this method are that it has a good effect on wrapping coal and sealing cracks, the material used is highly temperature-resistant, and the method has better applications to partial or small fires. Its disadvantages are also obvious; it comprises a small-medium flow, has a high cost, and exhibits colloid cracking after a long injection time; additionally, gel interactions with mine brine may produce poisonous gases [bib_ref] Characteristics and mechanism of prevention and control of coal spontaneous combustion with..., Ren [/bib_ref]. Three-phase foam injection: Three-phase foam has the following characteristics [bib_ref] Application of three-phase foam to fight an extraordinarily serious coal mine fire, Zhou [/bib_ref] : (a) it is formed by injecting nitrogen into a slurry, which leads to a substantial increase in the slurry volume; thus, the volume-expanding slurry forms a large range of coverage in a fire zone. (b) It encapsulates nitrogen so that it can stay in the fire zone longer. (c) It contains fly ash, mud and other solid substances that can provide it with long-term stability, thus allowing it to prevent coal oxidation. (d) Its foaming agent adds retardants that enable it to uniformly disperse "Five stages" division of coal spontaneous combustion and its countermeasures through coal and allow it to prevent the formation of coal-oxygen functional groups and the chain reaction of free radicals while also improving the wettability of the coal surface, thus greatly increasing the coal humidity. This method requires less investment and simple equipment; it is not difficult to operate, and it is characterized by continuous high-flow perfusion. Anti-fire material injection can quickly extinguish a fire and have better extinguishing effects, but it usually requires vast amounts (volumes) of original material for large fire zones, which is very expensive. Thus, this method is suitable for the evaporation stage and active stage because during these stages, fire rapidly develops and the production rates of CO, C 2 H 4 , C 2 H 6 and other harmful gases increase rapidly; thus, the fire must be quickly controlled. Sealing a fire zone. When a fire has been burning for more than 2 hours, it will be very large and thus difficult to directly extinguish. At this time, the fire zone should immediately be sealed [bib_ref] Study on control of disastrous open fires in underground coalmines, Singh [/bib_ref]. The essence of sealing a fire zone is reducing or even cutting off the oxygen supply to control or extinguish the coal combustion in a sealed fire zone. This method mainly involves building an airtight wall, which can not only form a sealed zone to cut off the oxygen but may also resist shock waves from gas explosions. Prior to building the airtight seals, one should judge the behavior of the fire based on the concentrations of fire indicator gases (CO, C 2 H 6 , CH 4 , C 2 H 4 , C 2 H 2 ) in the fire bundle tube monitoring system. When it is difficult to use direct extinguishment measures to control the fire, more preparations must be done, more human and material resources will be saved, and the fire will be controlled or even extinguished earlier. Therefore, the material preparations for building an airtight wall should be done when the spontaneous coal combustion reaches the active stage. ## Stage warning and disposal mechanisms of the spontaneous combustion of coal The risk of the spontaneous combustion of coal can be classified based on the following levels: very low risk, low risk, moderate risk, high risk, and extreme risk levels. These correspond to the successive five stages of the spontaneous combustion of coal. The warning levels are also divided into the following five sublevels: the blue, yellow, primary red, secondary red, and last red warning levels. Based on the analysis of the "five stages" division of the spontaneous combustion of coal and various anti-fire measures in mines, the stage warning and disposal table for the spontaneous combustion of coal is established and shown in [fig_ref] Table 3: Stage warning and disposal table of the spontaneous combustion of coal [/fig_ref]. Using the staged warning and disposal table can help workers carry out the targeted fire prevention measures based on the advance level of coal combustion. The prevention measures ascribed to various stages not only consider their practicality and effectiveness based on the stage characteristics but can also reduce their cost and impact on coal production. # Conclusions Based on the coal-oxygen-compound theory and due to the shortcomings of the "three stages" division of the phenomenon of the spontaneous combustion of coal, the "five stages" division was proposed; it includes the latent stage, heat accumulating stage, evaporation stage, active stage, and hypoxic stage. Experiments and calculations of the temperature corresponding to the zero activation energy of coal samples determined the critical point between the latent stage and the heat accumulation stage. The temperature of about 90˚C, at which the external coal moisture evaporates violently while the internal moisture has not yet evaporated, can be regarded as the critical point between the heat accumulating stage and the evaporation stage. The temperature of about 105˚C, at which the internal coal moisture evaporates completely, can be regarded as the ending point of the evaporation stage. After the dehydration of coal, its spontaneous combustion passes to the active stage; during this stage, the temperature of the coal and its related gaseous products increase dramatically. When the oxygen content drops down to 5%, the process enters the final, hypoxic stage. Our six-coal-sample experiments have shown that when the oxygen content is approximately 5%, the temperatures of all samples are above 170˚C; thus, the critical temperatures of the hypoxic stage of all six samples are beyond 170˚C, and the coal spontaneous combustion characteristics are greatly affected by the quality of the coal, thus affecting the lower thermal limit of the hypoxic stage. Based on the "five stages" division, the risk levels of the spontaneous combustion of coal are divided into the very low risk, low risk, moderate risk, high risk, and extreme risk levels, and the warning levels of the spontaneous combustion of coal are divided into the blue, yellow, primary red, secondary red, and last red warning levels. Considering the characteristics and applicability of the presented major prevention measures, the staged warning and disposal table for the spontaneous combustion of coal is established corresponding to the characteristics of each hazard and warning level. This work may provide guidance for preventing and controlling the spontaneous combustion of coal at different levels during the development of this process. ## Supporting information [fig] Fig 1: The small coal adiabatic oxidation testing system. 1-Nitrogen cylinder; 2-Oxygen cylinder; 3-Preheating pipeline; 4-Adiabatic tank; 5-Control panel; 6-Adiabatic oven; 7-Gas chromatograph; 8-Computer. https://doi.org/10.1371/journal.pone.0202724.g001 [/fig] [fig] Fig 2: The temperature-dependent activation energy curve.https://doi.org/10.1371/journal.pone.0202724.g002 "Five stages" division of coal spontaneous combustion and its countermeasures different moisture contents. Based on the data obtained, the temperature curves of coal samples with different moisture contents were drawn, as is shown inFig 3. [/fig] [fig] Fig 3: Thermal curves of temperatures of coal samples with different moisture contents. (A) The coal samples with water content of 5.6% and 7.2% were plotted together with original coal sample. (B) The coal samples with water content of 9.35%, 11.8%, 13.1%, and 15.25% were plotted together with the original coal sample. https://doi.org/10.1371/journal.pone.0202724.g003 "Five stages" division of coal spontaneous combustion and its countermeasures PLOS ONE | https://doi.org/10.1371/journal.pone.0202724 August 23, 2018 [/fig] [fig] Fig 4: Temperature-dependent variations in CO contents. https://doi.org/10.1371/journal.pone.0202724.g004 "Five stages" division of coal spontaneous combustion and its countermeasures [/fig] [fig] Fig 5: Temperature-dependent variations in C 2 H 4 contents. https://doi.org/10.1371/journal.pone.0202724.g005 [/fig] [fig] Fig 6: The relationship between oxygen concentration and temperature. https://doi.org/10.1371/journal.pone.0202724.g006 [/fig] [fig] S1: File. Data for Figs 2-6. (XLSX) [/fig] [table] Table 1: The industry index values of three coal samples. [/table] [table] Table 2: The proximate analysis test results. [/table] [table] Table 3: Stage warning and disposal table of the spontaneous combustion of coal. Measures I Heat production mainly relies on physical adsorption and chemical adsorption; oxidation reaction of coal transforms from passive to active. The latent stage Very low risk Blue warning Strengthen monitoring of the spontaneous combustion of coal. Temperature corresponding to zero activation energy of coal Temperature (about 90˚C) at which external moisture evaporates violently but internal moisture has not yet started to evaporate III The duration of this stage depends on the current coal moisture content. Seal the fire zone, drill to detect the fire area and inject anti-fire material. https://doi.org/10.1371/journal.pone.0202724.t003 "Five stages" division of coal spontaneous combustion and its countermeasures of the State Key Laboratory of coal Resources and Safe Mining (China University of Ming and Technology) (Grant SKLCRSM17KFA10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [/table]

Exploring the Public Health of Travel Behaviors in High-Speed Railway Environment during the COVID-19 Pandemic from the Perspective of Trip Chain: A Case Study of Beijing–Tianjin–Hebei Urban Agglomeration, China The outbreak and spreading of COVID-19 since early 2020 have dramatically impacted public health and the travel environment. However, most of the studies are devoted to travel behavior from the macro perspective. Meanwhile, few researchers pay attention to intercity travel behavior. Thus, this study explores the changes in the travel behavior of intercity high-speed railway travelers during the COVID-19 pandemic from the perspective of the individual. Using the smartphone data, this study first extracts the trip chains by proposing a novel method including three steps. The trip chain can describe the whole process of traveling, including individual characteristics, travel time, travel distance, travel mode, etc. Then, a Multinomial Logit model is applied to analyze the trip chains which verified the validity by using studentized residual error. The study finds that intercity travel behavior has changed in gender, age, travel mode choice, and travel purpose by comparing the trip chains between May 2019 and May 2021 in the Beijing-Tianjin-Hebei urban agglomeration. The method proposed in this study can be used to assess the impact of any long-term emergency on individual travel behavior. The findings proposed in this study are expected to guide public health management and travel environment improvement under the situation of normalized COVID-19 prevention and safety control. # Introduction A series of infectious diseases such as Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome Coronavirus (MERS), Ebola, etc., have seriously threatened people ranging from public health to psychological well-being. These contagions can also revolutionize people's lives in other aspects, for example through huge economic losses, travel environment, and education or career interruption [bib_ref] The impact of disease on family members: A critical aspect of medical..., Golics [/bib_ref] [bib_ref] The impact of COVID-19 on industry-related characteristics and risk contagion, Zhong-Fei [/bib_ref]. However, the COVID-19 pandemic, which broke out at the end of 2019 and quickly expanded at the beginning of 2020, has had more continuous and extensive influence on the entire mankind. By 14 November 2022, 631,935,687 cases and 6,588,850 deaths had been confirmed across the world [bib_ref] The relative impacts of disease on health status and capability well-being: A..., Mitchell [/bib_ref]. Contemporarily, COVID-19 is an enormous social risk that has formed tremendous threats to the worldon various aspects of society, including public health, environmental systems, aviation [bib_ref] A preliminary assessment of the impact of COVID-19 on environment-A case study..., Wang [/bib_ref] , agriculture, foreign trade [bib_ref] The effects of the COVID-19 pandemic on food losses in the agricultural..., Ilesanmi [/bib_ref] , political relations [bib_ref] Trade characteristics, competition patterns and COVID-19 related shock propagation in the global..., Wang [/bib_ref] , etc., ranging from people's mental health to social stability [bib_ref] The COVID-19 pandemic and the estrangement of US-China relations, Yang [/bib_ref]. Moreover, because the pandemic is very contagious and spreads through close contact with infected persons, COVID-19 has had a tremendous and unprecedented impact on daily life's behaviors, i.e., hosting, transportation, traveling, communication, etc. and the extent of the changes in travel behavior wrought during and after the pandemic remains unclear. In order to protect public health, a range of policies and measures have been proposed to control the virus around the world, such as social distancing, school closures and quarantining, severely restricting travel, and many other activities globally. Considering the human-to-human nature of COVID-19 transmission, urban lockdowns and travel restrictions are major policies to counter its spread. As the first country to discover coronavirus, China has launched a first-level emergency response to this public health emergency and rigidly enforced nonpharmaceutical interventions (NPIs) to constrain COVID-19 from 19 January 2020, to the present, including formulating a general strategy of "preventing the coronavirus from re-entering the country to cause a new epidemic" and the dynamic zero-COVID policy [bib_ref] The impact of COVID-19 vaccination for mental well-being, Kausik [/bib_ref]. Therefore, China has implanted a variety of more strict measures to make every possible effort to curb the spread of the virus, such as lockdowns, travel restrictions, and wearing masks in public spaces. Because of the strict NPIs, in China, the pandemic is now generally under control and the transport environment has returned to nearly normal. There have been 8,771,347 confirmed cases and 29,370 deaths reported, respectively 1.4% and 0.4% of the world level, while the population of China is nearly 17.7% of the global population by 14 November 2022. Currently, the continually mutating COVID-19 virus has induced a long-term period of recovery, i.e., the post-COVID-19 phase, which refers to the period of relative and regional stability after the initial uncontrolled global outbreak and growth of the pandemic, which has been referred to as the "new normal" by the WHO. Although the COVID-19 pandemic was well-controlled to some extent, these unprecedented measures have had a profound impact on the number and purposes of trips and modes of travel for a long time. Traffic flow has been restored gradually in different places, however, people's travel behavior is still affected by traffic control measures to different degrees. Intercity travel has more risks than traveling within a city in virus transmission because intercity travel increases the scope of people's communication to some extent [bib_ref] COVID-19 and its long-term effects on activity participation and travel behaviour: A..., Van Wee [/bib_ref] [bib_ref] Does density aggravate the COVID-19 pandemic? Early findings and lessons for planners, Hamidi [/bib_ref]. The roles of High-Speed Rail (HSR), aviation, and coach in the spread of COVID-19 have been investigated in China, and the presence of the HSR hub was significantly related to the pandemic diffusion. By 2019, more than 224 inland cities in China were connected by HSR, and the total mileage of HSR had reached 35,000 km, according to China's "Midto-Long-Term Railway Network Plan (Revised in 2016)" [bib_ref] Exploring the roles of high-speed train, air and coach services in the..., Zhang [/bib_ref]. With the development of HSR, the travel time between cities has been shortened, and commuting between urban agglomerations has gradually increased. Exploring the changes in the travel behavior of intercity High-speed railway travelers during the COVID-19 pandemic is very important for transportation policy formulation and sustainable development in the post-COVID-19 phase and normal daily context. In terms of COVID-19 impacts, some of the knowledge about travel behavior has been accumulated from early academic reviews, reflections, and visions that were based on theoretical reasoning or investigations of future traveler behavioral intentions [bib_ref] Impacts of intercity commuting on travel characteristics and urban performances in a..., Tao [/bib_ref]. Significant efforts have been devoted to investigating the impact of the COVID-19 pandemic on tourist arrivals or changes in travel behavior [bib_ref] How do Chinese tourists negotiate the constraints of engaging in post-COVID-19 domestic..., Zhang [/bib_ref] [bib_ref] Managing relationships in the Tourism Supply Chain to overcome epidemic outbreaks: The..., Gonzáalez [/bib_ref] [bib_ref] Tourism and COVID-19: Impacts and implications for advancing and resetting industry and..., Sigala [/bib_ref]. Most empirical studies pay attention to changes in travel frequency and mode choice, while few researchers focus on traveler characteristics and travel structure, both before and after the emergence of COVID-19. Meanwhile, massive objective traffic-related data are generally owned by governments and big corporations, and most scholars use questionnaires to explore the change in travel behavior, which limits the researchers to quantify the changes in individual behavior under the impact of COVID-19. Nowadays, with traveler behavior changes and the related effects resulting from the pandemic possibly being long-lasting, it is important to analyze the current situation and discuss the impacts of COVID-19 from the perspective of different individuals and groups. This study aims to explore the relationship between the impacts of the COVID-19 pandemic and changes in travel behavior in urban agglomeration from the perspective of individuals' whole travel procession, i.e., trip chain. Based on smartphone data which costs less and contains more information, this study proposes a novel method to identify intercity travelers using HSR and generate trip chains within Beijing-Tianjin-Hebei urban agglomeration. Then, a multinomial logit model is employed to reveal the changes in travel behavior by analyzing the intercity trip chains. To increase the persuasiveness of the results, smartphone data from the same month (May) in different years, which are the period before the outbreak of the pandemic and the post-COVID-19 phase, respectively, is used in this study. Understanding residents' trip chains provides critical support for various applications in public health, the transportation environment, and many other related fields. The results of this study can support public health management and travel environment improvement [bib_ref] The effect of COVID-19 and subsequent social distancing on travel behavior, De Vos [/bib_ref] [bib_ref] Modelling the impact of testing, contact tracing and household quarantine on second..., Martín-Corral [/bib_ref]. The rest of the paper is organized as follows: in the next section, a review is provided of the relevant literature. Section 3 presents an explanation of data processing. Section 4 describes the methodology. The empirical results are subsequently presented and discussed in Section 5. Finally, conclusions and recommendations for future works are outlined in Section 6. ## Literature review ## Impact of covid-19 on travel behavior A number of researchers have analyzed the impact of COVID-19 on the transportation system, such as the pandemic spread through transportation [bib_ref] Spatial transmission of COVID-19 via public and private transportation in China, Zheng [/bib_ref] , public transportation [bib_ref] COVID-19 spread and inter-county travel: Daily evidence from the U, Yilmazkuday [/bib_ref] [bib_ref] COVID-19 and Public Transportation: Current Assessment, Prospects, and Research Needs, Tirachini [/bib_ref] , travel demand [bib_ref] Impacts of COVID-19 on public transport ridership in Sweden: Analysis of ticket..., Jenelius [/bib_ref] [bib_ref] Modal share changes due to COVID-19: The case of Budapest, Bucsky [/bib_ref] , travel mode choice [bib_ref] The impact of COVID-19 on transport volume and freight capacity dynamics: An..., Loske [/bib_ref] , etc., after the outbreak of the pandemic, which indicates that people's travel behavior has changed under the threats of the virus and the related strict restrictions. Recognizing the underlying links between travel behavior and public health emergencies, researchers attempted to analyze the changes in travel behavior caused by COVID-19 [bib_ref] Modelling the effects of COVID-19 on travel mode choice behaviour in India, Bhaduri [/bib_ref]. Notably, changes in travel behavior are generally studied from the perspective of macroscopic and individual. A great part of the studies explored the changes in travel behavior from the macroscopic perspective using open statistics. Some researchers analyzed the impact of COVID-19 on population migration and urban traffic and found that while implementing antipandemic measures, the relevant government departments should concentrate more on densely inhabited and economically developed provinces and cities [bib_ref] Insights into the impact of COVID-19 on household travel and activities in..., Beck [/bib_ref]. Based on Baidu migration data, some researchers adopted a cluster analysis of origin and destination to analyze the relationship between migration and pandemic progression in the Guangdong-Hong Kong-Macao Greater Bay Area [bib_ref] An analysis of the domestic resumption of social production and life under..., Xu [/bib_ref]. It is believed that the degree of pandemic progression in the Guangdong-Hong Kong-Macao Greater Bay Area is positively correlated with the degree of immigration from Wuhan to the Greater Bay Area. The performance of the transportation system has been impeded to some extent under the influence of COVID-19 and the relevant policies. Although the transportation system has progressively recovered during the post-COVID-19 period [bib_ref] Transmission and control pressure analysis of the COVID-19 pandemic situation using multisource..., Wang [/bib_ref] , the operation of the transportation system is positively related to the spread of the pandemic [bib_ref] The relationship between trends in COVID-19 prevalence and traffic levels in South..., Lee [/bib_ref]. Due to the pandemic, in Spain, travel volume dropped dramatically while public transportation is extremely affected and private cars are relatively less affected [bib_ref] Analysis of road traffic pattern changes due to activity restrictions during COVID-19..., Patra [/bib_ref]. The amount of travel volume in the State of Qatar decreased by around 30% after the travel restrictions, while the distribution of all-day travel did not change significantly. Travel volume and distance have also obviously decreased in other countries, with about 55% of travel volume and 68% of distances traveled in the Netherlands, 90% of travel volume using public transportation, and 60% in daily distance traveled [bib_ref] Quantifying the impact of COVID-19 preventive measures on traffic in the State..., Muley [/bib_ref]. This type of study mainly pays attention to urban transportation systems employing various types of data to withdraw indicators to describe the characteristics of the urban transportation system, such as traffic volume, traffic structure, travel frequency, and travel mode choice [bib_ref] Quantifying the impact of COVID-19 on non-motorized transportation: A Bayesian structural time..., Zhang [/bib_ref]. A series of studies focuses on analyzing and simulating the changes in travel behavior from the perspective of the individual. Travel behavior of individuals is affected by differences in the gender, education level, lifestyle, travel purpose, etc., of travelers [bib_ref] Transport mode use during the COVID-19 lockdown period in Germany: The car..., Eisenmann [/bib_ref] [bib_ref] Impact of the COVID-19 pandemic on travel behavior in Istanbul: A panel..., Shakibaei [/bib_ref]. An online survey was employed in Tokyo, Japan, to explore the participation of urban residents in traveling during the early stage of COVID-19 [bib_ref] Influencing factors for potential bike-sharing users: An empirical analysis during the COVID-19..., Bergantino [/bib_ref]. It was found that people have voluntarily avoided leisure activities and dining outside since the early stage of the pandemic. The phenomenon of online activities such as online learning, working, shopping, etc., were adopted, and the change in transportation mode to walking and cycling are revealed through a preference-stated preference (RP-SP) survey in Chicago, USA, during the pandemic [bib_ref] Travel behavior changes during the COVID-19 pandemic in Japan: Analyzing the effects..., Parady [/bib_ref]. The number of travelers who aimed at education, visiting friends, and personal care decreased the most among all the purposes of travel in the Netherlands [bib_ref] How is COVID-19 reshaping activity-travel behavior? Evidence from a comprehensive survey in..., Shamshiripour [/bib_ref]. The frequency of travel for social, economic, and religious activities, in Nigeria, has considerably decreased based on a combination of data from active questionnaire surveys via email, social media, and professional networks [bib_ref] How COVID-19 and the Dutch 'intelligent lockdown' change activities, work and travel..., Haas [/bib_ref]. In several Chinese cities, i.e., Beijing, Shanghai, Guangzhou, Shenzhen, and Chongqing, the frequency of travel for shopping, tourism, eating out, and taking public transportation during the peak of the COVID-19 was significantly reduced, as compared to the period of outbreaking of the pandemic. Based on the license plate recognition data, the adjustment of travelers' behaviors under the influence of the pandemic was analyzed in Yiwu, Zhejiang Province, China [bib_ref] Impact of COVID-19 on transportation in Lagos, Mogaji [/bib_ref]. In China's Greater Bay Area, it was found that, in comparison to advantaged groups, socially disadvantaged groups experienced a steeper decline in travel mobility during the pandemic's peak, but a more significant recovery afterward by analyzing mobile phone data [bib_ref] Understanding travel behavior adjustment under COVID-19, Yao [/bib_ref]. This type of study mainly attempted to describe the changes in travel behavior from the individual perspective through questionnaire data, mobile phone data, and so on. In order to analyze the changes in travel behavior more comprehensively and accurately, this study explores the changes in the travel behavior of intercity high-speed railway travelers during the COVID-19 pandemic from the perspective of the individual. Additionally, a novel method to generate trip chains of intercity High-speed railway travelers, which includes identification of intercity travelers, extraction of travel features within the city, and generation of intercity trip chains, is first proposed in this study. ## Smartphone-based trip chain For decades, numerous researchers have explored the character of people's travel behavior, which is measured as daily trip frequency, trip purposes, departure time, travel duration, travel distance, travel modes, trip sequences, trip destinations, travel companions, etc., which can be displayed in trip chains, to provide long-term guidance and short-term strategies for urban planning and transportation development [bib_ref] Analyzing COVID-19's impact on the travel mobility of various social groups in..., Pan [/bib_ref] [bib_ref] An Empirical Study of Travel Time Variability and Travel Choice Behavior, Jackson [/bib_ref] [bib_ref] Examining Trip-Chaining Behavior: Comparison of Travel by Men and Women, Mcguckin [/bib_ref] [bib_ref] Travel time variability: A review of theoretical and empirical issues, Noland [/bib_ref] [bib_ref] Model of Household Trip-Chain Sequencing in Emergency Evacuation, Murray-Tuite [/bib_ref] [bib_ref] Modeling constrained destination choice for shopping: A GIS-based, time-geographic approach, Scott [/bib_ref] [bib_ref] Does flexi time affect departure time choice for morning homebased commuting trips?..., He [/bib_ref] [bib_ref] Will you escort your child to school? The effect of spatial and..., He [/bib_ref] [bib_ref] Activity-travel behavior research: Conceptual issues, state of the art, and emerging perspectives..., Buliung [/bib_ref]. A trip chain is a collection of interconnected trips that begin at one origin, pass through one or more stops, and then end at a destination. It was first used to test the influence of pre-planning activities on a certain day in the trip chain on the traffic pattern of a day from the perspective of utility maximization in 1979 [bib_ref] Zooming into individuals to understand the collective: A review of trajectory-based travel..., Yue [/bib_ref]. Traditionally, data used in trip chains were mainly obtained from travel surveys. Travel surveys are important data for analyzing and evaluating travel behavior daily. A typical household travel survey is meant to collect extensive information about travel and activity behavior from a sample of houses or people. Nevertheless, a city-wide household travel survey is often costly to collect, lacks instantaneity and continuity, short survey duration, has incomplete information, and quickly becomes out-of-date [bib_ref] A theoretical and empirical model of trip chaining behavior, Adler [/bib_ref] [bib_ref] Elimination of the Travel Diary: Experiment to Derive Trip Purpose from Global..., Wolf [/bib_ref] [bib_ref] National Household Travel Survey: A look into the travel patterns of older..., Collia [/bib_ref]. With the development of technology, Global Positioning System (GPS) is used in travel surveys. The first use of GPS in a travel survey was in Austin, Texas, in 1996, when 200 families were requested to describe their trip chains through computer-assisted telephone interviews while concurrently employing GPS in their vehicles to track vehicular trips [bib_ref] Critical factors for active transportation to school among low-in-come and minority students:..., Mcdonald [/bib_ref]. Due to vehicle GPS loggers only collecting vehicular trips and having technical issues, such as a cold start signal acquisition delay, there are obvious limitations for vehicle GPS loggers to collect travel by all modes [bib_ref] Trip reporting in household travel diaries: A comparison to GPS-collected data, Casas [/bib_ref]. With the use of wearable GPS devices, travel surveys are getting more attention because they can collect higher-quality data by continually and correctly capturing respondents' travel paths without introducing hassles to respondents [bib_ref] Comparative Analysis of Global Positioning System-Based and Travel Survey-Based Data, Bricka [/bib_ref]. However, limitations still exist which restrict GPS use in travel surveys, for example, the price of GPS devices is expensive and the fully productive response of GPS devices is low. Recently, with the advancement of information and communication technologies, many kinds of novel data, like smartphone data, have been used to trip chain research. Smartphone data can not only capture the real-time location information of travelers, but also describe the attributes of individuals, such as age, gender, destination type, and so on. Compared with traditional survey data and GPS data, smartphone data have several clear advantages, such as lower costs, more locational sensor data sources, more accurate data, wider geographic area, more coverage of population, and greater likelihood for respondents to be collecting data at all times throughout the survey period, which attract scholars in various fields to apply them to travel behavior research, and a certain amount of progress has been made to date [bib_ref] Extracting Activity-travel Diaries from GPS Data: Towards Integrated Semi-automatic Imputation, Feng [/bib_ref]. Some studies employed smartphones to passively collect GPS data with minimum user interaction or input on the app [bib_ref] Mobile phone data from GSM networks for traffic parameter and urban spatial..., Steenbruggen [/bib_ref] , while few researchers make efforts on extracting trip chains from smartphone data. In order to overcome the above limitations, this study generated intercity trip chains with smartphone data which are from May of 2019 and 2021. ## Data description and processing ## Study area In this study, the travel behavior of travelers who use the high-speed railway for intercity travel between Beijing, Tianjin, Shijiazhuang, and Handan, within the Beijing-Tianjin-Hebei urban agglomeration, as shown in [fig_ref] Figure 1: Study area [/fig_ref] , is investigated. Beijing-Tianjin-Hebei urban agglomeration, which is a capital economic circle and the center of the political and cultural center, plays an important role in the development of China. It is also an essential part of the Chinese transportation system because it is one of the four important international terminal clusters in the guidelines of China's comprehensive national transport network. Beijing, Tianjin, Shijiazhuang, and Handan are the core cities in the Beijing-Tianjin-Hebei urban agglomeration which have the largest and most representative inter-city traffic volume in the urban agglomeration. The data period of this study is May 2019 and May 2021, which are before the outbreak of COVID-19 and the period of the post-COVID-19 phase, respectively. In China, May includes a five days' vacation which is the Labor Day holiday and does not includes long vacation, such as winter vacation and summer vacation. In addition, the climate in May is relatively comfortable for traveling. Therefore, the data in May is representative to some extent. Since April 2020, Chinese residents resumed travel, and the tourism industry was gradually recovering [bib_ref] Smartphone app versus GPS Logger: A comparative study, Stopher [/bib_ref]. China has entered into a special and unique recovering period that is distinct from the other countries that were still undergoing serious impact from COVID-19. ## Smartphone data The raw data used in this study is smartphone data, and it is collected from the platform of Data-as-a-Service (DAAS) which is operated by one of the Chinese communication operators with a 30% market share within the Beijing-Tianjin-Hebei urban agglomeration. The raw data has several kinds of information as follows. The first type of data is basic location data which includes the location information of mobile communication base stations, the grid where the base stations are located, the road node, the latitude and longitude coordinates of the base stations, etc. The second type of data is residence data. According to the system setting, a user's stay point is supposed to be a location where he stays for more than 30 min, which includes the start and end time of presence, location, frequency of presence, etc. The location data in this study follow the principle of World Geodetic System 1984 (WGS84). The third type of data is travel data which includes travel start and end time, travel location, travel speed, travel time, travel through base stations and road nodes, etc. The fourth type of data is user attribute data which stores basic personal information, internet service records, and other related information. The fifth type of data is coding data which stores the user's ID card domicile location, internet access behavior label, location, and other information. This study collects a grand total of 27,120,000 attribute information of smartphone users and 54,900,000,000 travel data, as shown in [fig_ref] Table 1: The data size. [/fig_ref]. ## Data processing Due to the generation mechanism of smartphone data and the data collection system, there will be invalid data and noise data such as duplicate, missing, and wrong data in the raw data, which will generate an adverse effect on extracting trip chains. First, a clustering algorithm of k-means was used to clean the noise data, which were erroneous or abnormal, such as unknown gender, unknown age, etc.The software of Statistical Package for the Social Sciences (SPSS) was used to complete the k-means algorithm. The raw data was imported to SPSS in step 1. The method of iteration and classification were chosen in step 2. The number of iterations were set by 10, while the convergence conditions default to 0 in step 3. Then, the k-means algorithm was completed and the results were showen in SPSS. The noise data which were erroneous or abnormal were cleaned. Secondly, in order to facilitate the analysis of results, this study discretizes the variables of age, distance and time as shown in [fig_ref] Table 2: Variable Discretization [/fig_ref]. Lastly, a series of variables, such as road number, smartphone brand, internet traffic, etc., which are not related to generating trip chains are removed, while a total of 15 variables are selected as shown in [fig_ref] Table 3: Data description. [/fig_ref]. # Methodology ## Trip chain generation The trip chain of urban agglomeration is not only a simple process for individuals to reach the ending point from the starting point. It also contains a lot of information, such as travel time, space trajectory, mode of transportation, and so on, which are interrelated and interact with each other and are accompanied by the whole process of travel. Travel purpose and mode choice are simultaneously decided by travelers [bib_ref] Autonomous shuttle bus service timetabling and vehicle scheduling using skip-stop tactic, Cao [/bib_ref] [bib_ref] Exploring Traffic Crash Occurrence Mechanism toward Cross-Area Freeways via an Improved Data..., Yang [/bib_ref]. In this study, a trip chain refers to a whole process of intercity travel which includes travel within the origin city, intercity travel by using a high-speed railway, and travel within the destination city as shown in [fig_ref] Figure 2: Schematic diagram of urban agglomeration trip chain [/fig_ref]. The method to generate trip chains is as follows. In this study, as the data of the DAAS platform shows, the user stay data is extracted intelligently in the unit of the city. Therefore, in order to identify intercity travelers, we use the following method: Step 1: Generate a hub layer based on the geographic location of the research hub, convert it into a wkt file, and import it into the DAAS platform; Step 2: Spatial matching, if the user stays in the space range of the research hub, then extract the user's data as the hub-staying user data set; Step 3: Data grouping: group the data set according to users, hubs, and dates to obtain the time series of each user's stay in each research hub every day; Step 4: Travel generation: extract the user's last stop at the current hub and the first stop at the next hub, the travel between the two stops is the user's intercity travel between the hubs. The technical route of this part is as shown in [fig_ref] Figure 3: Extraction of intercity travel OD [/fig_ref] : ## Extraction of travel feature within the city This study uses a two-layer clustering algorithm based on time and space to identify the user's travel stop point. The algorithm is divided into two stages. In the first stage, we use the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm to cluster user trajectory data spatially. Then, we perform time clustering on the result data of the first stage, and finally, accurately identify the user's staying point, and then obtain the user's travel OD. The specific steps of this algorithm are as follows: Step 1: Extract all trajectory data of relevant users in the research area; Step 2: Through all the trajectory data of each user and input them as data; Step 3: Determine the parameters of the DBSCAN algorithm, including the minimum number of stay points (MinP) and the minimum distance of stay points (MinR); Step 4: The DBSCAN model calculates and outputs the stay points of each user; Step 5: Sort the stay points in time series, and calculate the stay time of each cluster; Step 6: Determine whether the stay time of each cluster is greater than the time threshold T. If it is not, it indicates that the residence time is not matched to the stay point; Step 7: Output a set of stay points based on the identified stay points and connect them to form the travel OD of each user. The technical route of this part is as shown in [fig_ref] Figure 4: Travel OD extraction algorithm based on spatio-temporal clustering [/fig_ref] : In this algorithm, it is necessary to adjust and verify the MinP and the MinR in the DBSCAN algorithm, and T in time clustering, or set it based on experience. In this study, we determined the MinP as 8 according to the average number of daily trajectory points of the user, and then we compared the results of identification under different MinR and T. According to the 5th Beijing Comprehensive Transportation Survey (2014), the average trip frequency of the residents is 2.75. We input different threshold values into our OD algorithm and then compare the calculated results with the above survey data. It can be found from [fig_ref] Table 4: The results of the average trip frequency under different thresholds [/fig_ref] that the trip frequency is closest to the travel survey result when T is set as 30 min and MinR is set as 500 m. Additionally, this also indicates that the result of our OD algorithm is consistent with the actual survey data, which proves the accuracy of the algorithm. ## Generation of intercity trip chains Travelers may have several trips within origin city and destination city, but we only need the trips that include hub A and hub B. Therefore, after extracting the OD of each user's intercity travel and the user's intercity travel OD between the research hubs, we match the user's entire trip chains between two cities by filtering the trips which included hub A and hub B in the same day. The rules are as follows: Step 1: The same user on the same day: Step 2: The user's travel destination of the current trip in city A is the research hub; Step 3: The user's next trip will be an intercity trip, and the destination of the trip will be the research hub of city B; Step 4: The user takes the research hub of city B as the starting point to generate trips in city B. The process of generating individual trip trains is shown in [fig_ref] Figure 5: The process of generating individual trip trains [/fig_ref] : ## Validity test of trip chains The data used in this study has the characteristics of large size, high dimension, and discretization. Therefore, the existing mainstream outlier detection methods such as simple box plots cannot do anomaly detection for categorical data [bib_ref] Novel Coupling-Decoupling Strategy for Scheduling Autonomous Public Transport Vehicles in Over-crowded Corridors, Cao [/bib_ref] [bib_ref] Identification of dynamic traffic crash risk for cross-area freeways based on statistical..., Yang [/bib_ref] [bib_ref] Real-time schedule adjustments for autonomous public transport vehicles, Cao [/bib_ref] [bib_ref] Predicting Freeway Traffic Crash Severity Using XGBoost-Bayesian Network Model with Consideration of..., Yang [/bib_ref]. Under the assumption that travel time increases with travel distance, the two variables of travel distance and travel time (in fact, a total of four variables, divided into the departure process and arrival process) can be jointly detected (multivariate outlier detection, the joint interaction of multiple variables to detect anomalous data records.). Therefore, the studentized residual error of least squares linear regression model is used to identify abnormal values. The mathematical formulation can be characterized as follows: [formula] y = β 0 + β 1 x + e(1) [/formula] Here, the exploratory variable x is travel distance while the explained variable y is travel time. β 0 is the intercept and β 1 is the gradient. e is the random error term which is set in linear regression model. The least square method is used to fit the model and estimate the parameters. The parameter estimation formula is as follows: [formula] β 1 = ∑ n i=1 y i x i − (∑ n i=1 y i )(∑ n i=1 x i ) n ∑ n i=1 x 2 i − (∑ n i=1 x i ) 2 n (2) β 0 = y −β 1 x(3) [/formula] Here, x i and y i are, respectively the explanatory variable and explained variable of the ith data. n represents the total number of the data. y is the arithmetic mean of the explained variable, while x is the arithmetic mean of the explanatory variable.β andβ 1 are, respectively the least squares estimate of β 0 and β 1 . The fitting model is as follows: [formula] y =β 0 +β 1 x(4) [/formula] The ordinary residual of each data is as follows: [formula] e i = y i −ŷ i = y i − β 0 +β 1 x i(5) [/formula] Next, let y i denote the observed response of the i th observation in the dataset, and y i_del denote the predicted response of the ith observation using the model fitted to the dataset after the i th observation has been removed. The i th deleted residual is defined as: [formula] d i = y i −ŷ i_del(6) [/formula] The studentized residual t i for each observation is then calculated by dividing its deletion residual by its estimated standard deviation: [formula] t i = d i s(d i ) = e i MSE (i) (1 − h ii ) ∼ t(n − p − 1)(7) [/formula] This turns out to be equivalent to the ordinary residual divided by a factor that includes the mean square error based on the estimated model with the i th observation deleted, MSE(i) (refers to the mean square error of the fitted model after removing i th observation) and the leverage, h ii (refers to the diagonal elements of the "hat matrix", which corresponds to the i th observation). In addition, t i obeys a T-distribution with n − p − 1 degrees of freedom and 95% confidence level, n refers to the dataset size and p refers to the number of parameters of the fitted model. ## Multinomial logit model A trip chain is a record of the whole process of a traveler which includes age, gender, mode choice, travel purpose, travel distance, travel time, and so on. In this study, a multinomial logit regression model with trip chains as the dependent variable is constructed to explore changes in travel behavior. Firstly, a utility function is introduced as follows: [formula] T ij = x i β j + ε ij (i = 1, 2, . . . , n)(8) [/formula] where T ij is the utility of individual i choosing trip chain j. x i is a set of individual attributes and travel characteristics while β j is the parameter vector to be estimated. ε ij denotes the error term. The probability of individual i choosing trip chain j is as follows: [formula] P i (j) = P T ij > T im , (m = j)(9) [/formula] When ε ij follows the generalized extreme value distribution, the model formula is as follows: [formula] P i (j) = exp x i β j ∑ J m=1 exp(x i β m )(10) [/formula] # Results and discussion ## Generation and validity test of trip chains Based on the dataset, the current study then used the mathematical programming software Python 3.5 on an Acer laptop with Intel Core i5-10210U 1.60 GHz 2.11 GHz CPU and 12 GB RAM to generate trip chains. There were 614,285 pieces of trip chains generated, which include 351,340 pieces of trip chains in May 2019 and 262,945 pieces of trip chains in May 2021. Next, the method of studentized residual was applied to test the validity of the trip chains. Firstly, the studentized residuals were calculated through simulating the model with the variables of destination travel distance and time, which was used to analyze the abnormal data points. The results are shown in [fig_ref] Table 5: Identification of abnormal values based on the variables of destination travel distance... [/fig_ref] , and. In [fig_ref] Table 5: Identification of abnormal values based on the variables of destination travel distance... [/fig_ref] , the data whose travel distance from hub B to destination (DTD) are not match the travel time from hub B to destination (DTT) are deleted. For example, in the first data of [fig_ref] Table 5: Identification of abnormal values based on the variables of destination travel distance... [/fig_ref] , the value of DTD is "11" which equals 50-55 min, while the value of DTT is "212" which equals 1055-1060 km. In this situation, the travel speed is 1152-1271 km/h which is extremely abnormal.shows the results of identification of abnormal value based on the variables of destination travel distance and time. The red data points are qualified data while the blue data points are abnormal data. Then, the anomalous value was deleted from the data set of trip chains. Based on the updated set of trip chains, the variables of origin travel distance, and time were used to detect the abnormal value. The results are shown in Tables 7 and 8, and. In Tables 7 and 8, the data whose travel distance from origin to hub A (OTD) are not match the travel time from origin to hub A (OTT) are deleted. For example, in the first data of [fig_ref] Table 7: Identification of abnormal values based on the variables of origin travel distance... [/fig_ref] , the value of OTD is "13" which equals 60-65 min, while the value of OTT is "252" which equals 1255-1260 km. In this situation, the travel speed is 1167-1255 km/h which is extremely abnormal.shows the results of identification of abnormal value based on the variables of origin travel distance and time. The red data points are qualified data while the blue data points are abnormal data. There was a total of 574,221 pieces of trip chain obtained after the validity test. The number of trip chains was 327,877 with removing 9950 trip chains in the origin process and 13,513 trip chains in the destination process, respectively from the data set of May 2019. The number of trip chains was 246,344 with removing 1545 trip chains in the origin process and 15,056 trip chains in the destination process, respectively, from the data set of May 2021. ## Results of multinomial logit model The dependent variable of the multinomial logit model is Y which is the combination of travel mode. The independent variables of the model are shown in [fig_ref] Table 9: The independent variables of the model [/fig_ref]. The age of the user The groups are shown in [fig_ref] Table 2: Variable Discretization [/fig_ref] Travel_DS_O The travel distance from the origin to hub A The groups are shown in [fig_ref] Table 2: Variable Discretization [/fig_ref] Travel_Wt_O The waiting time in hub A The groups are shown in [fig_ref] Table 2: Variable Discretization [/fig_ref] Travel_Wt_D The waiting time in hub B The groups are shown in [fig_ref] Table 2: Variable Discretization [/fig_ref] Travel_DS_D The travel distance from hub B to the destination The groups are shown in [fig_ref] Table 2: Variable Discretization [/fig_ref] 8 Activity_type The origin type and destination type 1: Residence 2: Work 0: Visit According to the input requirements of the stats model package, the independent variable Y, i.e., the combination of travel mode, was transformed to values 0, 0.2, 0.4, 0.6, 0.8, and 1 which, respectively represent private car-HSR-private car, private car-HSR-railway, private car-HSR-public transportation, railway-HSR-private car, public transportation-HSR-private car and public transportation-HSR-railway. The dependent variables were also transformed. Male was the reference of the variable of gender while the value of female was transformed to [T.2]. The remaining dependent variables were transformed as the same. Therefore, the results of the model are shown in Tables 10-12. The parameter with white color indicates OR = 1, the parameter with red color indicates OR < 1, and the parameter with green color indicates OR > 1. [fig_ref] Table 1: The data size. [/fig_ref] - From the perspective of gender, the coefficient of C (Gender) [T.2] is negative in all modes, which indicates that women are more likely to use the private car-HSR-private car trip chain for travel, followed by private car-HSR-public transportation. When Y equals 1, the coefficient of C(Gender) [T.2] is smallest in all modes which indicates that women refuse to travel by public transportation-HSR-private car a lot. The reason is probably that women usually travel more from developing cities to developed cities. # Discussion ## Analysis of travel behavior in may 2019 from ## - Weekdays have a positive effect on the division of the travel mode combination of private car-HSR-railway and private car-HSR-public transportation, which indicates that travelers are more likely to arrive at the hub by private car. It is because more business trips are generated on weekdays and travelers tend to choose comfortable travel modes, while travelers prefer economic travel mode on weekends. - Most age groups tend to travel through private car-HSR-railway. Moreover, travelers whose ages are from 19 to 39 are also likely to choose to travel between intercity without a private car, and middle-aged people travel more often compared to other age groups [bib_ref] A Parallel FP-Growth Mining Algorithm with Load Balancing Constraints for Traffic Crash..., Yang [/bib_ref]. Individuals whose age is over 60 do not choose public transportation to travel within the intercity, which is opposite within the city. ## - Travel distance has a significant in travel mode choice. Travelers prefer to travel by public transportation when travel distance is short. - Considering the waiting time at the starting city transportation hub, overall, as the waiting time increases, the willingness of the traveler to choose this trip chain starts decreasing. A comparison between trip chains shows that travelers arriving at a transportation hub by railway are more likely to accept longer waiting times. The influence of whether the travel day is a weekday or not is largely consistent between May 2019 and May 2021. The change is that the predictive effect of weekday travel on the railway-HSR-private car chain changes from a negative effect to a positive effect. Meanwhile, its predictive effect on private car-HSR-railway and public transportation-HSR-private car increases, suggesting that travelers prefer a private car with less exposure risk of COVID-19 for weekday travel. There is previous study found that more people prefer to use private cars and bikes instead of public transportation modes during the pandemic [bib_ref] Predicting travel attitudes among university faculty after 9/11, Staats [/bib_ref]. Before the COVID-19 outbreak, car use was more limited because of its high environmental and social costs, including pollution, obesity, energy consumption, and other negative externalities [bib_ref] The impacts of COVID-19 on travel behavior and initial perception of public..., Nezir [/bib_ref]. In China, highway tolls were suspended from 17 February 2020 to 6 May 2020 by the Ministry of Transport of the People's Republic of China, to encourage people to travel by car during the pandemic [bib_ref] Active transportation and physical activity: Opportunities for collaboration on transportation and public..., Sallis [/bib_ref]. ## - The prediction of age characteristics on the trip chain changes mainly in the public transportation-HSR-railway trip chain, and the predictive effects of the age group above 39 years old for this trip chain all change from negative to positive during the post-COVID-19 phase. ## - The effect of travel distance within the origin city in travel mode is similar in 2019 and 2021 with a slight difference in that the positive effect diminishes and the negative effect increases in the travel mode of public transportation -HSR-private car and public transportation-HSR-railway. The result is consistent with the previous study that for longer distances, people shifted from public transport to private car [bib_ref] Measuring changes in travel behavior pattern due to COVID-19 in a developing..., Abdullah [/bib_ref]. It is probably because COVID-19 leads to travelers preferring private cars instead of public transportation due to the threats of infection. This finding can also be seen in the effect of traveler's waiting time at the starting city transportation hub, compared to 2019, the positive effect of waiting time on public transportation (Y = 0.8, Y = 1) predicting relative to other modes of travel decreases in the year 21. There has not been a significant difference in the influence of travel distance and waiting time in the process of traveling within destination cities between 2019 and 2021. - Compared with the period pre-outbreak of the pandemic, intercity travelers who travel from home have an increasingly positive effect on the travel modes of the private car during the post-COVID-19 phase. Individuals also tend to temporarily restrict travel for daily social and economic activities or use relatively low-risk modes of transportation to avoid contracting infectious diseases which is consistent with previous studies [bib_ref] Choice behavior of commuters' rail transit mode during the COVID-19 pandemic based..., Tan [/bib_ref] [bib_ref] A paradigm shift in urban mobility: Policy insights from travel before and..., Thombre [/bib_ref]. [fig_ref] Table 1: The data size. [/fig_ref] - The travel behavior of intercity high-speed railway travelers during the COVID-19 pandemic has changed significantly by analyzing the model parameters of 2019 and 2021. In the odds ratios results of the Multinomial Logit model as shown in [fig_ref] Table 1: The data size. [/fig_ref] , the parameters of white, red, and green, respectively, represent odds ratios equal to 1, odds ratios are less than 1 and odds ratios are greater than 1. It is obvious that the odds ratios of the same independent variables and dependent variables vary between the years 2019 and 2021. Studies found that COVID-19 reduces intercity travel directly and indirectly by influencing industry development and transport connectivity [bib_ref] Exploring the dynamic impacts of COVID-19 on intercity travel in China, Tao [/bib_ref]. ## Comparison analysis of travel ## - In the aspect of travel mode choice, travelers tend to choose the trip chain of public transportation-HSR-public transportation in 2019 while travelers tend to choose the trip chain of private car-HSR-public transportation in 2021. The spread of COVID-19 has decreased the willingness to choose public transport where travelers are more likely to be infected due to intensive passenger flow. On weekdays, the mode choice of the private car is significantly increasing in 2021. ## - Depending on the variable of C (Activity_type), it is found that the willingness to use a private car for trips on the home-based side is increasing with the value of odds ratios increasing by about 20%. - However, the odds ratios value of variable C (Travel_Wt_O) decreased by about 40% in the groups of railway-HSR-private car and public transportation during the post-COVID-19 phase, which indicates that the travel volume transferred from public transportation to private car. - Compared to the period pre-outbreak of the pandemic, intercity High-speed railway travelers are more willing to travel by private car, which has less risk, during the post-COVID-19 phase [bib_ref] Role of latent factors and public policies in travel decisions under COVID-19..., Chen [/bib_ref]. The nature of risk aversion encourages people to use private vehicles instead of public transportation. Interestingly, however, this characteristic was not reflected by gender and age, as female travelers were less likely to use a private car to reach transportation hubs in 2021, which is consistent with the finding that a higher proportion of male travelers transferred to the private car than female travelers after the outbreak in some studies. # Conclusions The transportation environment can reflect the status of urban vitality and economic development, whereas the analysis of travel behavior adjustments under COVID-19 and the corresponding influencing factors can help to deepen the understanding of how the pandemic has affected the daily life of individuals, thus helping to analyze the far-reaching influence of the pandemic on public health and society. Meanwhile, analyzing the impact of the pandemic on the transportation system and individual behavior can provide support for public health management and travel environment improvement during the post-COVID-19 phase. Based on smartphone data, a novel method to generate Spatio-temporal trip chains of intercity high-speed railway travelers, which includes identification of intercity travelers, extraction of travel features within the city, and generation of intercity trip chains is first proposed in this study. Next, the studentized residual method is used to test the validity of the generated trip chains. By the combination of the two methods, a total of 574,221 pieces of trip chain are obtained. Then, a Multinomial Logit model is employed to explore the changes in the travel behavior of intercity High-speed railway travelers during the COVID-19 pandemic from the perspective of a trip chain with A case study of Beijing-Tianjin-Hebei urban agglomeration, China. This study finds a series of changes in travel behavior from the individual perspective between the period pre-outbreak of COVID-19 and the post-COVID-19 phase. These methods and findings can help us understand travel behavior from the individual perspective and provide critical support for the formulation of public health policy and the improvement of the transportation environment during the post-COVID-19 phase. The contributions of the present study are threefold: (1) The method of generating intercity trip trains possesses excellent applicability which can be directly used in other urban agglomerations. (2) It includes both individual attributes and travel characteristics and explicitly examines the changes in intercity travel behavior. Most previous studies largely focused on changing travel patterns and travel frequency during COVID-19. (3) The findings proposed in this study are expected to guide public health management and travel environment improvement under the situation of normalized COVID-19 prevention and safety control. There are also some limitations in this study. First, due to the limitation of the data, this study can only analyze and compare the changes in travel behavior between May 2019 and May 2021, while the characteristics of travel behavior are different in the other 11 months. More data is warranted to further compare the results of this study. On the one hand, we plan to purchase more data by applying for funding in the future. On the other hand, we will actively seek cooperation with the communication operator to jointly study this issue. Second, we explored the changes in the travel behavior of intercity high-speed railway travelers in the context of the whole Beijing-Tianjin-Hebei urban agglomeration. In the future, we can further compare the changes in travel behavior on an urban scale. Meanwhile, we can also compare the travel behavior in Beijing-Tianjin-Hebei urban agglomeration with other urban agglomeration, such as China's Greater Bay Area, the Yangtze River Delta urban agglomeration, and so on. [fig] Figure 1: Study area: Beijing-Tianjin-Hebei urban agglomeration. [/fig] [fig] Figure 2: Schematic diagram of urban agglomeration trip chain. 4.1.1. Identification of Intercity Traveler [/fig] [fig] Figure 3: Extraction of intercity travel OD. [/fig] [fig] Figure 4: Travel OD extraction algorithm based on spatio-temporal clustering. [/fig] [fig] Figure 5: The process of generating individual trip trains. [/fig] [fig] Figure 6: (a) Identification of abnormal value based on the variables of destination travel distance and time (May 2019); (b) Identification of abnormal value based on the variables of destination travel distance and time (May 2021). [/fig] [fig] Figure 7: (a) Identification of abnormal value based on the variables of origin travel distance and time (May 2019); (b) Identification of abnormal value based on the variables of origin travel distance and time (May 2021). [/fig] [fig] Author: Contributions: Methodology, S.Y.; Project administration, B.L.; Supervision, B.L. and D.L.; Writing-original draft, S.Y.; Writing-review and editing, S.Y.; Proofreading, S.Y. All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This research was funded by the Basic Scientific Research Business Expenses Special Funds from National Treasury (Grant No. 2021-9059b, 2021-9059a), and the National Key Research and Development Program of China (Grant No. 2018YTB1601300). [/fig] [table] Table 1: The data size. [/table] [table] Table 2: Variable Discretization. [/table] [table] Table 3: Data description. [/table] [table] Table 4: The results of the average trip frequency under different thresholds. [/table] [table] Table 5: Identification of abnormal values based on the variables of destination travel distance and time (May 2019).Wd denotes character of the day. OTM denotes the travel mode from origin to hub A. OTD denotes the travel distance from origin to hub A. OTT denotes the travel time from origin to hub A. DTM denotes the travel mode from hub B to destination. DTD denotes the travel distance from hub B to destination. DTT denotes the travel time from hub B to destination. The last row represents that the following abnormal values are omitted due to space limitation which is the same inTables 6-8. [/table] [table] Table 6: Identification of abnormal value based on the variables of destination travel distance and time (May 2021). [/table] [table] Table 7: Identification of abnormal values based on the variables of origin travel distance and time (May 2019). [/table] [table] Table 9: The independent variables of the model. [/table]

Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis Background:The MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants.Results: A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C-and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis.Conclusion: A comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis identified several MYBs with potential role in development and stress response of plants. # Background Transcription factors are essential regulators of gene transcription and usually consist of at least two domains namely a DNA-binding and an activation/repression domain, that function together to regulate the target gene expression. The MYB (myeloblastosis) transcription factor family is present in all eukaryotes. "Oncogene" v-MYB was the first MYB gene identified in avian myeloblastosis virus. Three v-MYB-related genes namely c-MYB, A-MYB and B-MYB were subsequently identified in many vertebrates and implicated in the regulation of cell proliferation, differentiation, and apoptosis. Homologous genes were also identified in insects, fungi and slime molds. A homolog of mammalian c-MYB gene, Zea mays C1, involved in regulation of anthocyanin biosynthesis, was the first MYB gene to be characterized in plants. Interestingly, plants encode large number of MYB genes as compared to fungi and animals. MYB proteins contain a MYB DNA-binding domain, which is approximately 52 amino acid residues in length, and forms a helix-turn-helix fold with three regularly spaced tryptophan residues. The three-dimensional structure of the MYB domain showed that the DNA recognition site α-helix interacts with the major groove of DNA. However, amino acid sequences outside the MYB domain are highly divergent. Based on the number of adjacent MYB repeats, MYB transcription factors are classified into four major groups, namely 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB containing one, two, three and four MYB repeats, respectively. In animals, R1R2R3type MYB domain proteins are predominant, while in plants, the R2R3-type MYB domain proteins are more prevalent. The plant R2R3-MYB genes probably evolved from an R1R2R3-MYB gene progenitor through loss of R1 repeat or from an R1-MYB gene through duplication of R1 repeat. In plants, MYB transcription factors play a key role in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. Several R2R3-MYB genes are involved in regulating responses to environmental stresses such as drought, salt, and cold. Transgenic rice over expressing OsMYB3R-2 exhibited enhanced cold tolerance as well as increased cell mitotic index. Enhanced freezing stress tolerance was observed in Arabidopsis over-expressing OsMYB4. Arabidopsis AtMYB96, an R2R3-type MYB transcription factor, regulates drought stress response by integrating ABA and auxin signals. Transgenic Arabidopsis expressing AtMYB15 exhibited hypersensitivity to exogenous ABA and improved tolerance to drought, and cold stress. The AtMYB15 negatively regulated the expression of CBF genes and conferred freezing tolerance in Arabidopsis. Other functions of MYBs include control of cellular morphogenesis, regulation of secondary metabolism, meristem formation and the cell cycle regulation. Recent studies have shown that the MYB genes are posttranscriptionally regulated by microRNAs; for instance, AtMYB33, AtMYB35, AtMYB65 and AtMYB101 genes involved in anther or pollen development are targeted by miR159 family. MYB TF family genes have been identified in a number of monocot and dicot plants, and evolutionary relationship between rice and Arabidopsis MYB proteins has been reported. We report here genome-wide classification of 155 and 197 MYB TF family genes in rice and Arabidopsis, respectively. We also analysed abiotic stress responsive and tissue specific expression pattern of the selected MYB genes. To map the evolutionary relationship among MYB family members, phylogenetic trees were constructed for both rice and Arabidopsis MYB proteins. Several over-represented cis-regulatory motifs in the promoter region of the MYB genes were also identified. # Results and discussion ## Identification, classification and structural analysis of myb family members Genome-wide analysis led to the identification of 155 and 197 MYB genes in rice and Arabidopsis, respectively, with their mapping on different chromosomes (Additional file 1:. We used previously assigned names to the MYB genes; for instance, AtMYB0 (GL1) name was accepted for the first identified R2R3 MYB gene; subsequently identified R2R3 MYB genes were named as AtMYB1, AtMYB2, etc. in Arabidopsis. We classified MYB transcription factors in to four distinct groups namely "MYB-related genes", "MYB-R2R3", "MYB-R1R2R3", and "Atypical MYB genes" based on the presence of one, two, three and four MYB repeats, respectively. Our analysis revealed that the MYB-R2R3 subfamily consisted of the highest number of MYB genes, with 56.77 and 70.05% of the total MYB genes in rice and Arabidopsis, respectively , b). In the R2R3-MYB proteins, N-terminal consists of MYB domains, while the regulatory C-terminal region is highly variable. Presence of a single MYB-like domain (e.g. hTRF1/ hTRF2) in their C terminus is required for telomeric DNA binding in vitro. Earlier study revealed that the R2R3-MYB related proteins arose after loss of the sequences encoding R1 in an ancestral 3R-MYB gene during plant evolution. In contrast, only few MYB-R1R2R3 genes were identified in Arabidopsis and rice with 5 and 4 genes, respectively. The category "MYBrelated genes" usually but not always contain a single MYB domain. We found that "MYB-related genes" represented 40 and 26.39% of the total MYB genes in rice and Arabidopsis, respectively , b), and thus constituted the second largest group of MYB proteins in both rice and Arabidopsis. We also identified one MYB protein in rice and two MYB proteins in Arabidopsis that contained more than three MYB repeats and these belong to "Atypical MYB genes" group. The AT1G09770 in Arabidopsis and LOC_Os07g04700 in rice have five MYB domains and are called as CDC5-type protein, whereas AT3G18100 of Arabidopsis has four MYB domains and is named as 4R-type MYB; Additional file 1:. The 4R-MYB proteins belong to the smallest class, which contains R1/R2-like repeats. MYB genes can also be classified into several subgroups based on gene function, such as Circadian Clock Associated1 (CCA1) and Late Elongated Hypocotyl (LHY), Triptychon (TRY) and Caprice (CPC). CPC and TRY belong to the R3-MYB group and are mainly involved in epidermal cell differentiation, together with ENHANCER OF TRY AND CPC1, 2 and 3 (ETC1, ETC2 and ETC3), and TRICHOMELESS1 and 2 (TCL1 and TCL2). Here, we observed that CCA1, CPC and LHY subgroups contain 23, 3 and 1 'MYB-related' TF, respectively in Arabidopsis. To further understand the nature of MYB proteins, their physiochemical properties were also analyzed. The MYB proteins have similar grand average hydropathy (GRAVY) scores. Kyte and Doolittleproposed that higher average hydropathy score of a protein indicates physiochemical property of an integral membrane protein, while a negative score indicates soluble nature of the protein. We observed that all MYB proteins in rice and Arabidopsis, except AT1G35516 had a negative GRAVY score, suggesting that MYBs are soluble proteins, a character that is necessary for transcription factors. Minimum and maximum score of GRAVY were recorded as −1.287 (LOC_Os02g47744) and −0.178 (LOC_Os08g37970) in rice, and −1.359 Chromosome-wise distribution of MYB transcription factor genes. a) rice, b) Arabidopsis. We classified MYB transcription factors in to four distinct groups namely "MYB-related genes", "MYB-R2R3", "MYB-R1R2R3", and "Atypical MYB genes" based on the presence of one, two, three and four MYB repeats, respectively. (AT5G41020) and 0.612 (AT1G35516) in Arabidopsis, respectively. We also calculated average isoelectric point (pI) value. The mean pI values for MYB-1R, R2R3 and R1R2R3 protein families were 7.55, 6.90 and 7.25 in rice, and 7.55, 6.89 and 6.80 in Arabidopsis, respectively. The average molecular weight of MYB-1R, R2R3 and R1R2R3 protein families were 31.128, 34.561 and 72.52 kDa in rice, and 34.186, 35.875 and 86.217 kDa in Arabidopsis, respectively (Additional file 1:. ## Functional classification of myb transcription factors MYB proteins perform wide diversity of functions in plants. The R2R3-MYB proteins are involved in plant specific processes, such as control of secondary metabolism or cellular morphogenesis. Gene ontology (GO) analysis suggested that R2R3-MYB genes, namely AtMYB16, AtMYB35, AtMYB5/AtMYB80, and AtMYB91 may regulate cell, anther, trichome and leaf morphogenesis, respectively. Likewise, R2R3-type genes, namely OsMYB16, OsMYB88, OsMYB117, LOC_Os01g50110 and LOC_Os03g38210 may regulate morphogenesis in rice. In addition to R2R3-type MYBs, two MYB-related genes, LOC_Os01g43180 and LOC_Os09g23200 may also regulate morphogenesis in rice. R2R3-type AtMYB10 and AT2G47210, MYB-related AT3G09600, and R1R2R3type AtMYB3R4 genes were identified with GO function, such as N-terminal protein myristoylation, histone H3 acetylation, and regulation of DNA endoreduplication, respectively. Previous studies have shown that genes encoding 3R-MYB proteins have regulatory role in cell cycle control. We also found that AtMYB3R4 may be involved in cell cycle control (GO: 0007049). GO analysis of MYB proteins illustrated that 98.70% OsMYB and 98.47% AtMYB were fully involved in transcription activation, while rest of the MYB proteins were classified in to other GO functions, such as kinase activity, protein binding, transcription repressor activity, etc. GO analysis categorized rice LOC_Os01g62660 as signal transducer (GO: 0004871) and transcription activator. The R2R3type AtMYB4 was classified into transcriptional repressor group. The AtMYB4 expression is down regulated by exposure to UV-B light, indicating that derepression of its target genes is an important mechanism for acclimation to UV-B in Arabidopsis. In our study, AtMYB34; a R2R3-type MYB protein, has been found with catalytickinase as well as transcription activator molecular functions as reported earlier. The AtMYB34 is also involved in defense response against insects. In consistent with previous report, AtMYB23 was found to have protein binding (i.e. interaction with GL3) as well as DNA-binding functions. The subcellular localization of MYB proteins was predicted using several localization predictor softwares. The predicted locations of the MYB proteins were also verified by gene ontology under keyword "GO cellular component" and species-specific localization prediction tools, e.g., AtSubP for Arabidopsisto enhance the accuracy of prediction. Consensus outcome revealed that 98.71% OsMYB and all AtMYB proteins were found to be nuclear localized and confirmed by the presence of nuclear localization signal (NLS). The remaining two members of MYB proteins in rice were predicted to be localized in mitochondria and plasma membrane. A Complete list of functional assignment of MYB genes is given in Additional file 2:. ## Gene structure and intron distribution To understand the structural components of MYB genes, their exon and intron organization was analyzed. We observed that 17 (10.96%) OsMYB and 9 (4.56%) AtMYB genes were intronless, which is in conformity with the previous analysis. To identify conserved intronless MYB genes, blastall (BLASTP) was performed between protein sequence of all the predicted intronless genes of rice and Arabidopsis, and vice versa. Expected cut-off value of 1e-6 or less was used to identify the conserved intronless genes. We found that 13 (76.47%) and 7 (77.77%) intronless OsMYB and AtMYB genes, respectively, were orthologs. Other intronless MYB genes that fulfilled the matching criteria, expected cut-off value of 1e-10 or less were referred to as paralogs. We observed that 4 (23.52%) and 2 (22.22%) intronless OsMYB and AtMYB genes, respectively, were paralogs (Additional file 3:. This analysis showed that intronless genes of rice and Arabidopsis are highly conserved, and may be involved in similar regulatory functions in these plants. To explore the intron density in MYB genes with introns, we divided ORF into three zones, namely N-terminal, central and C-terminal zones. We observed that mid region had high density of introns, i.e., 43.99 and 50.63% in rice and Arabidopsis, respectively. The number of introns per ORF varied, with maximum of 12 and 15 introns in OsMYB4R1 and AT2G47210, respectively. Rice LOC_Os01g43180 and Arabidopsis AT3G10585 genes contain shortest introns with 37 and 43nt, respectively. Among all MYB genes, LOC_Os08g25799 of rice and AT1G35515 of Arabidopsis contained longest intron with an intron length of 5116 and 1621nt, respectively (Additional file 4: . In order to gain insight into exon-intron architecture, the intron positions on MYB domains were investigated. In support with previous results, we also noticed that a large number of rice (26.45%) and Arabidopsis (38.57%) R2R3-type domain containing proteins have a conserved splicing pattern with three exons and two introns. However, some R2R3-type MYB genes lack one intron either in R2 or R3 repeat in rice (23.22%) and Arabidopsis (25.88%). It has been proposed that the duplication of R2 in an early form of two repeat MYB proteins gave rise to the R1R2R3 MYB domains. Hence, we also investigated the exon-intron structure of R1R2R3-type MYB proteins. We observed that 3R-MYB proteins contained conserved three exons-two introns pattern in R1 and R2 and one conserved intron in R3 repeat in Arabidopsis. Similarly, in rice, three out of five 3R-MYB genes have similar structure; Additional file 4: . These results indicate similar distribution of introns in MYB domain in both rice and Arabidopsis. ## Chromosomal distribution, tandem repeats and duplication The position of all 155 OsMYB and 197 AtMYB genes were mapped on chromosome pseudomolecules available at MSU (release 5) for rice and TAIR (release 8) for Arabidopsis. The distribution and density of the MYB genes on chromosomes were not uniform. Some chromosomes and chromosomal regions have high density of the MYB genes than other regions. Rice chromosome 1 and Arabidopsis chromosome 5 contained highest density of MYB genes, i.e. 21.93 and 28.93%, respectively. Conversely, chromosome 11 of rice and chromosome 2 of Arabidopsis contained lowest density of MYB genes, i.e. 2.58 and 12.69%, respectively. Distribution of MYB genes on chromosomes revealed that lower arm of chromosomes are rich in MYB genes, i.e. 65.16% in rice and 52.79% in Arabidopsis. Distribution pattern also revealed that chromosome 5 in rice, and chromosome 2 and 5 in Arabidopsis contained higher number of MYB genes with introns, i.e. 29.41 and 33.33%, respectively. Intronless MYB genes are absent in chromosome 4, 9, 10, 11 and 12 in rice, and chromosome 1 in Arabidopsis. Distribution of MYB genes on chromosomal loci revealed that 11 (7.09%) in rice and 20 (10.15%) genes in Arabidopsis were found in tandem repeats suggesting local duplication. Chromosome 6 in rice and chromosome 1 in Arabidopsis contained higher number of tandem repeats, i.e. 7 genes and showed over-representation of MYB genes. Three direct tandem repeats were found on chromosome 6 (LOC_Os06g07640; LOC_Os06g07650; LOC_Os06g07660) in rice, and chromosome 1 (AT1G66370, AT1G66380; AT1G66390) as well as chromosome 5 (AT5G40330; AT5G40350; AT5G40360) in Arabidopsis. Four direct tandem repeats were also observed on chromosome 3 (AT3G10580, AT3G10585, AT3G10590 and AT3G10595) in Arabidopsis. Manual inspection unraveled 44 (28.38 %) and 69 (35.02%) homologous pairs of MYB genes in rice and Arabidopsis, respectively evolved due to segmental duplication. We also observed that two homologous pairs in Arabidopsis contained one MYB gene and other than that was not classified as MYB gene in TAIR (release 10) databases. About 44 (28.39%) OsMYB and 69 (35.02%) AtMYB genes showed homology with multiple genes including MYB genes from various locations on different chromosomes. It is widely accepted that redundant duplicated genes will be lost from the genome due to random mutation and loss of function, except when neo-or sub-functionalization occur.suggested that gene duplications in R2R3-type MYB family occurred during earlier period of evolution in land plants. Recently, a range of duplicated pair of MYB genes in R2R3-type protein family has been identified in maize. Among the tandem repeat pair (AT2G26950 and AT2G26960) in Arabidopsis, AtMYB104 (AT2G26950) is down-regulated by ABA, anoxia and cold stress, but up-regulated under drought, high temperature and salt, while AtMYB81 (AT2G26960) expression pattern was opposite to that of AtMYB104, i.e., AtMYB81 is up-regulated in response to ABA, anoxia and cold stress, but down regulated under drought, high temperature and salt stresses. Similar diversification was also observed in the duplicate pair (LOC_Os10g33810 and LOC_Os02g41510) in rice. OsMYB15 (LOC_Os10g33810) expressed in leaf, while LOC_Os02g41510 expressed in shoot and panicle tissue. These spatial and temporal differences among different MYB genes evolved by duplication indicate their functional diversification. ## Cis-motifs in the myb gene promoters Discovery of regulatory cis-elements in the promoter regions is essential to understand the spatial and temporal expression pattern of MYB genes. Co-expressed genes may be regulated by a common set of transcription factors, and can be detected by the occurrence of specific cis-regulatory motifs in the promoter region. Hence, we analyzed the promoter regions of the drought up-and down-regulated MYB genes identified from our previous microarray data experiments. Among the top five cis-motifs identified by this analysis, only CCA1 (TTWKTTWWTTTT) was the previously known cismotif. Although, CCA1 cis-motif was reported as common feature of rice genome, we found CCA1 cis-motif only in genes that are down-regulated by drought stress. The CCA1 motif was found in 94.74% of the drought down-regulated genes in rice. Furthermore, we investigated the group of R2R3-type MYB genes for the discovery of gene-specific new cis-regulatory element in both rice and Arabidopsis. Likewise, we discovered novel cis-motifs with no description in PLACE database, except for CCA1 motif in rice. The CCA1 motif was found in 70.45% of the R2R3-type MYB genes in rice. The CCA1, a MYB-related TF, binds to CCA1 motif and regulate circadian clock controlled expression of genes in Arabidopsis. To validate our prediction, we examined the diurnal or circadian clock controlled MYB expression using "Diurnal Version 2.0". About 47.74 and 90.86% MYB genes were found to be diurnal/circadian-regulated in rice and Arabidopsis, respectively (Additional file 5: . Noticeably, we did not find any common motif between rice and Arabidopsis MYB promoter regions, indicating divergence in regulatory region of MYB genes between monocot and dicot species. ## Expression of myb genes under abiotic stresses To identify MYB genes with a potential role in abiotic stress response of plants, we analyzed the expression . In our previous microarray data experiments, we found that 142 (92.26%) MYB genes were expressed in seedlings of rice (Additional file 8: Previous studies have shown that over-expression of MYB genes improved abiotic stress tolerance of rice and Arabidopsis. In addition to these, we have identified additional MYB genes that are regulated by drought The coding sequence were aligned using BLAST 2 SEQUENCES to quantitate the sequence differences between the gene pairs. AtMYB genes were up regulated in cold, drought and salt stress, respectively (Additional file 9:, b and c, Additional file 10:. We analyzed expression patterns of 60 OsMYB and 21 AtMYB genes using QRT-PCR. These genes were selected based on phylogenetic analysis and one gene from each cluster was selected for expression analysis. Out of the 60 genes examined by QRT-PCR, 28 OsMYB genes were up-regulated (≥ 1.5 fold change) under drought stress in rice cv. Nagina 22. We also found that LOC_Os02g47744, LOC_Os12g41920 and LOC_Os06g19980 were highly up-regulated (≥ 4 fold change), indicating their potential role in drought stress. QRT-PCR analysis of 21 MYB genes in Arabidopsis revealed that 7 AtMYB genes were up-regulated (≥ 1.5 fold changes) and another 7 AtMYB genes were downregulated (≤ 1.5 fold change) under drought stress. ## Tissue-specific expression In rice, a tissue breakdown of EST evidence for MYB genes was analyzed using the Rice Gene Expression Anatomy Viewer, MSU database. In case of Arabidopsis, tissue-specific expressions of MYB genes were obtained from GENEVESTIGATOR tool. The expression patterns of MYB genes in different tissues are listed in Additional file 11: . The results showed that large numbers of OsMYB genes (32.90%) were highly expressed in the panicle, leaf and shoots (Additional file 12:. EST frequency analysis suggested that OsMYB genes, LOC_Os02g34630, LOC_Os08g05510, LOC_Os01g74590, LOC_Os02g09480, LOC_Os09g36730, OsMYB4, LOC_Os10g41200 and LOC_Os01g13740 are highly expressed in flower, anther, endosperm, pistil, shoot, panicle, immature seed and whole plant, respectively. In case of leaves, we observed that three MYB genes, i.e., OsMYB48, LOC_Os06g40710 and LOC_Os10g41200 showed highest levels of expression. In Arabidopsis, the following MYB genes expressed at a very high level: AtMYBCDC5 in callus and seed; AT1G19000 in seedling and stem; AT1G74840 in root and root tip; AT1G26580 in flower, AtMYB91 in shoot, and AtMYB44 in pedicel and leaves. In wheat, TaMYB1 showed high expression in root, sheath and leaf, while TaMYB2 expression was highest in root and leaf, but at low in sheath. TaMYB1 and TaMYB2 showed a very high sequence similarity with AtMYB44 and OsMYB48, respectively. Our analysis also revealed that these two MYBs are highly expressed in leaf as in case of wheat. These analyses will be useful in selecting candidate genes for functional analysis of their role in a specific tissue. ## Evolutionary relationship To understand the evolutionary relationship among MYB family genes, phylogenetic trees were constructed using the multiple sequence alignment of MYB proteins. The tree revealed that tandem repeat and homologous pairs were grouped together into single clade with very strong bootstrap support (Additional file 13:. These results further support gene duplication in rice and Arabidopsis during evolution which may allow functional diversification by adaptive protein structures. It was also noticed that few "homologues pairs" (e.g. AT5G16600-AT3G02940 in Arabidopsis; LOC_Os12g07610-LOC_Os12g07640 in rice) and "tandem repeat pairs" (e.g. AT3G12720-AT3G12730 in Arabidopsis; LOC_Os06g14700-LOC_Os06g14710 in rice) were found in distinct clade, indicating that only few members had common ancestral origin that existed before the divergence of monocot and dicot. MYB proteins from rice and Arabidopsis with same number of MYB domains were grouped into a single clade. For instance, all the MYBs belonging to R1R2R3 family in both rice and Arabidopsis were clustered into single clade. Within the R2R3 clade, MYBs from rice and Arabidopsis were not found in distinct groups. These results suggest that significant expansion of R2R3-type MYB genes in plants occurred before the divergence of monocots and dicots, which in agreement with the previous studies. Finally, we observed that two CDC5type and one 4-repeat MYB orthologs were clustered into single clade and might have been derived from an ancient paralog of widely distributed R2R3 MYB genes. # Conclusions Our study provides genome-wide comparative analysis of MYB TF family gene organization, sequence diversity and expression pattern in rice and Arabidopsis. Structural analysis revealed that introns are highly conserved in the central region of the gene, and R2R3-type MYB proteins usually have two introns at conserved positions. Analysis of length and splicing of the intron/exon and their position in MYB domain suggested that introns were highly conserved within the same subfamily. Most of the MYB genes are present as duplicate genes in both rice and Arabidopsis. Phylogenetic analysis of rice and Arabidopsis MYB proteins showed that tandem repeat and homologous pair was grouped together into single clade. Consensus motif analysis of 1kb upstream region of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. The comparative analysis of MYB genes in rice and Arabidopsis elucidated chromosomal location, gene structure and phylogenetic relationships, and expression analysis led to the identification of abiotic stress responsive and tissue-specific expression pattern of the selected MYB genes, suggesting functional diversification. Our comprehensive analyses will help design experiments for functional validation of their precise role in plant development and stress responses. # Methods ## Identification of myb gene family in rice and arabidopsis To identify MYB transcription factor family genes, we searched and obtained genes annotated as MYB in MSU (release 5) for rice and TAIR (release 8) for Arabidopsis by using in-house PERL script along with careful manual inspection. The primary search disclosed 161 and 199 members annotated as "MYB" or "MYB-related genes" in MSU and TAIR database, respectively. We observed that some protein members lack MYB-DNA binding domain but still annotated as MYB protein family in MSU and TAIR database. We discarded these proteins based in the annotation in MSU (release 7) for rice and TAIR (release 10). Finally, we obtained 155 and 197 MYB genes in rice and Arabidopsis, respectively. The gene identifiers were assigned to each OsMYB and AtMYB genes to avoid confusion when multiple names are used for same gene. Uncharacterized MYB genes are denoted here by their locus id. ## Myb annotation To identify number of domains present in MYB protein we executed domain search by Conserved Domains Database(http://www.ncbi.nlm.nih.gov/Structure/ cdd/cdd.shtml) and pfam database(http://pfam. sanger.ac.uk/)with both local and global search strategy and expectation cut off (E value) 1.0 was set as the threshold for significance. Only significant domain found in rice and Arabidopsis MYB protein sequence were considered as a valid domain. To get more information about nature of the MYB protein, grand average of hydropathy (GRAVY), PI and the molecular weight were predicted by ProtParam tool available on Expert Protein Analysis System (ExPASy) proteomics server (http:// www.expasy.ch/tools/protparam.html). The subcellular localization of MYB proteins were predicted by Protein Localization Server (PLOC) (http://www.genome.jp/SIT/ plocdir/), Subcellular Localization Prediction of Eukaryotic Proteins (SubLoc V 1.0) (http://www.bioinfo.tsinghua.edu. cn/SubLoc/eu_predict.htm), SVM based server ESLpred (http://www.imtech.res.in/raghava/eslpred/submit.html), and ProtComp 9.0 server (http://linux1.softberry.com/ berry.phtml?topic=protcomppl&group=programs&subgroup= proloc). Further, species-specific localization prediction system was utilized for Arabidopsis (AtSubP, http:// bioinfo3.noble.org/AtSubP/). MYB protein function in term of their Gene Ontology (GO) was predicted by GO annotation search page available at MSU (http:// rice.plantbiology.msu.edu/downloads_gad.shtml) and TAIR (http://www.arabidopsis.org/tools/bulk/go/index.jsp) for rice and Arabidopsis, respectively. Localization consensus was predicted based on majority of result. The confidence level was acquired by assigning equal numeric value (e.g. one) to each general localization predictor and higher value to gene ontology (e.g. two) and species specific predictor (e.g. three). ## Identification of over-represented motifs We discovered over represented cis-motif consensus pattern in 1 kb upstream sequence from translational initiation codon of MYB genes in both rice and Arabidopsis using the Multiple Expectation maximization for Motif Elicitation analysis tool(MEME version 4.1.0, http:// meme.sdsc.edu/meme/meme-intro.html). This program was used to search best 5 cis-motif consensus patterns of 8-12 bases width, with E-value < 0.01, only on the forward strand of the input sequences. Motifs graph were plotted according to their position within the region using WebLogo tool (http://weblogo.berkeley.edu/logo. cgi). Discovered motifs were analyzed using PLACE(http://www.dna.affrc.go.jp/PLACE/). Diurnal and circadian controlled MYB expression was explored from "Diurnal Version 2.0" (Mockler lab; http://diurnal.mocklerlab.org/). # Phylogenetic analysis To generate the phylogenetic trees of MYB transcription factor family genes, multiple sequence alignment of MYB protein sequence were performed using COBALT program(http://www.ncbi.nlm.nih.gov/tools/cobalt/). COBALT program automatically utilize information about bona fide proteins (i.e. MYB domains in this case) to execute multiple sequence alignment and build phylogenetic tree. The dendrogram were constructed with the following parameters; method-fast minimum evolution, max sequence difference-0.85, distance-grishin (protein). ## Myb localization, tandem repeat and duplication To map the gene loci on rice and Arabidopsis chromosomes pseudomolecules were used in MapChart (version 2.2) programfor rice and chromosome map toolfor Arabidopsis available on The Arabidopsis Information Resource (TAIR) database (http://www. arabidopsis.org/jsp/ChromosomeMap/tool.jsp). Tandem repeats were identified by manual visualization of rice and Arabidopsis physical map. Duplication or homologous pair genes were obtained by the segmental genome duplication segment (http://rice.plantbiology.msu. edu/segmental_dup/) and Arabidopsis Syntenic Pairs / Annotation Viewer (http://synteny.cnr.berkeley.edu/AtCNS/) in rice (distance = 500kb) and Arabidopsis, respectively. The tandem repeat and homologous pairs were aligned with the BLAST 2 SEQUENCE tool available on National Center on Biotechnology Information (NCBI) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi/). # Gene structure analysis To know more about intron / exon structure, MYB coding sequence (CDS) were aligned with their corresponding genomic sequences using spidey tool available on NCBI (http://www.ncbi.nlm.nih.gov/spidey/). To identify conserved intronless genes between rice and Arabidopsis, local protein blast (BLASTP) (http://www.molbiol. ox.ac.uk/analysis_tools/BLAST/BLAST_blastall.shtml) was performed for protein sequences of all predicted intronless genes in rice against all predicted intronless gene in Arabidopsis, and vice versa. Hits with 1e-6 or less were treated as conserved intronless genes and hits with 1e-10 or less were treated as paralogs. The cutoff of sequence identity was considered as ≥ 20% over the 70% average query coverage. # Expression analysis Expression support for each gene model is explored through gene expression evidence search page (http://rice. plantbiology.msu.edu/locus_expression_evidence.shtml) available at MSU for rice and GENEVESTIGATOR tool (https://www.genevestigator.com/) for Arabidopsis. MYB genes for which no ESTs were found, blast (BLASTP and TBLASTN) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search using NCBI databases was performed. Significant similarity of MYB genes with MYB genes of other plant species was searched. To measure the MYB expression level in abiotic stress plant QTLGE database was used (http://www.scbit. org/qtl2gene/new/) for rice and GENEVESTIGATOR tool (https://www.genevestigator.com/) for Arabidopsis. To identify tissue specific expression level of OsMYB genes in rice, highly expressed gene search (http://Rice. plantbiology.msu.edu/tissue.expression.shtml) available at MSU were used. For Arabidopsis, GENEVESTIGATOR tool (https://www.genevestigator.com/gv/user/gvLogin.jsp) was used. ## Plant materials and growth conditions The plant materials used were drought tolerant rice (Oryza sativa L. subsp. Indica) cv. Nagina 22 and Arabidopsis thaliana ecotype Columbia. The seeds were surface sterilized. Rice seeds were placed on absorbent cotton, which was soaked overnight in water and kept in medium size plastic trays. Arabidopsis seeds were germinated on MS-agar medium containing 1% Sucrose and seven days old seedlings were transferred to soilrite for further growth. The rice and Arabidopsis seedlings were grown in a greenhouse under the photoperiod of 16/8 h light/dark cycle at 28°C ± 1 and 23°C ± 1, respectively. ## Drought stress treatment Drought was imposed to 3-weeks old rice seedlingsand 5-week-old Arabidopsis plants by withholding water till visible leaf rolling was observed. Control plants were irrigated with sufficient water. Plant water status was quantified by measuring relative water content of leaf. Control plants showed 96.89 and 97.49% RWC (relative water content), while stressed plants showed.86 and 65.2% RWC in rice and Arabidopsis, respectively. ## Real-time rt-pcr Total RNA from rice and Arabidopsis were isolated by TRIzol Reagent (Ambion) and treated with DNase (QIAGEN, GmbH). The first strand cDNA of rice and Arabidopsis was synthesized using Superscript III Kit (Invitrogen) from 1 μg of total RNA according to manufacturer's protocol. Reverse transcription reaction was carried out at 44°C for 60 min followed by 92°C for 10 min. Five ng of cDNA was used as template in a 20 μL RT reaction mixture. Sixty three pairs of rice and 51 pairs of Arabidopsis gene specific primers were used to study expression of MYB transcription factor. Gene specific primers were designed using IDT PrimerQuest (http://www. idtdna.com/scitools/applications/primerquest/default.aspx). Ubiquitin and actin primers were used as an internal control in rice and Arabidopsis, respectively. The primer combinations used here for real-time RT-PCR analysis specifically amplified only one desired band. The dissociation curve testing was carried out for each primer pair showing only one melting temperature. The RT-PCR reactions were carried out at 95°C for 5 min followed by 40 cycles of 95°C for 15s and 60°C for 30s each by the method described previously by . For qRT-PCR, QuantiFast SYBR Green PCR master mix (QIAGEN GmbH) was used according to manufacturer's instruction. The threshold cycles (C T ) of each test target were averaged for triplicate reactions, and the values were normalized according to the C T of the control products (Os-actin or Ubiquitin) in case of rice and Arabidopsis, respectively. MYB TFs expression data were normalized by subtracting the mean reference gene CT value from individual CT values of corresponding target genes (ΔCT). The fold change value was calculated using the expression, where ΔΔCT represents difference between the ΔCT condition of interest and ΔCT control. The primer sets used to study the MYB TFs expression profile are given in the Additional file 14: .

The Metabotropic Purinergic P2Y Receptor Family as Novel Drug Target in Epilepsy Epilepsy encompasses a heterogeneous group of neurological syndromes which are characterized by recurrent seizures affecting over 60 million people worldwide. Current anti-epileptic drugs (AEDs) are mainly designed to target ion channels and/or GABA or glutamate receptors. Despite recent advances in drug development, however, pharmacoresistance in epilepsy remains as high as 30%, suggesting the need for the development of new AEDs with a non-classical mechanism of action. Neuroinflammation is increasingly recognized as one of the key players in seizure generation and in the maintenance of the epileptic phenotype. Consequently, targeting signaling molecules involved in inflammatory processes may represent new avenues to improve treatment in epilepsy. Nucleotides such as adenosine-5 -triphosphate (ATP) and uridine-5 -triphosphate (UTP) are released in the brain into the extracellular space during pathological conditions such as increased neuronal firing or cell death. Once released, these nucleotides bind to and activate specific purinergic receptors termed P2 receptors where they mediate the release of gliotransmitters and drive neuronal hyperexcitation and neuroinflammatory processes. This includes the fast acting ionotropic P2X channels and slower-acting G-protein-coupled P2Y receptors. While the expression and function of P2X receptors has been well-established in experimental models of epilepsy, emerging evidence is now also suggesting a prominent role for the P2Y receptor subfamily in seizure generation and the maintenance of epilepsy. In this review we discuss data supporting a role for the P2Y receptor family in epilepsy and the most recent finding demonstrating their involvement during seizure-induced pathology and in epilepsy. # Introduction The primary treatment for epilepsy is the use of anti-epileptic drugs (AEDs). These drugs control seizures by shifting the balance of inhibitory and excitatory drive in the brain. 30% of patients, however, are pharmacoresistant to all available AEDs and between 40 and 50% of patients on AEDs suffer adverse effects [bib_ref] Quality of life of people with epilepsy: a European study, Baker [/bib_ref]. Current major goals of epilepsy research are to develop treatment strategies that impact upon disease emergence and progression, show efficacy within the currently pharmacoresistant cohort and have a lower burden of adverse effects. To this end, the role of neuroinflammation in icto-and epileptogenesis is receiving growing attention [bib_ref] Inflammation and Epilepsy: preclinical findings and potential clinical translation, Terrone [/bib_ref]. Purinergic signaling provides a mechanism by which hyperexcitation can lead to an inflammatory response and whereby inflammation can lead to hyperexcited networks. As such, the targeting of purinergic signaling is a promising strategy for developing new treatment options . Purinergic signaling is mediated via two families of purinergic receptors: ionotropic P2X receptors and metabotropic P2Y receptors [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref] , both receptor subtypes responding to extracellular adenine or uridine nucleotides. While much of the focus of purinergic signaling in epilepsy has focussed on the P2X receptor family, the role of P2Y receptors in epilepsy has, to date, received much less attention [bib_ref] Purinergic signaling in epilepsy, Rassendren [/bib_ref] [bib_ref] The ATP-gated P2X7 receptor as a target for the treatment of drug-resistant..., Beamer [/bib_ref]. In this review, we summarize data from the emerging field and suggest directions in which P2Y research in epilepsy should develop. ## Seizures, status epilepticus, and epilepsy Seizures are a transient symptom resulting from abnormally excessive or synchronous neuronal firing in the brain [bib_ref] ILAE official report: a practical clinical definition of epilepsy, Fisher [/bib_ref]. In general, seizures do not last longer than 1-2 min and are self-limiting [bib_ref] How long do most seizures last? A systematic comparison of seizures recorded..., Jenssen [/bib_ref]. Prolonged or recurrent seizures without intervening recovery periods, however, are classified as status epilepticus, a medical emergency [bib_ref] Status epilepticus in adults, Betjemann [/bib_ref]. Beyond epilepsy, seizures can have many etiologies, including acute insults, such as fever, hypoxia, low blood sugar, brain tumors, lack of sleep, substance abuse, or traumatic brain injury (TBI). Seizures can be classified according to their etiology, semiology, and anatomical focus [bib_ref] Classifications of seizures and epilepsies, where are we? -A brief historical review..., Chang [/bib_ref]. The transition from seizures to status epilepticus is often due to a failure of endogenous anticonvulsant mechanisms, such as the internalization or desensitization of γ-aminobutyric acid (GABA) A receptors [bib_ref] GABA synapses and the rapid loss of inhibition to dentate gyrus granule..., Naylor [/bib_ref] [bib_ref] Status epilepticus in adults, Betjemann [/bib_ref]. Status epilepticus is the second most common neurological emergency behind stroke, with an annual incidence of 10-41 cases per 100,000 [bib_ref] Risk of unprovoked seizure after acute symptomatic seizure: effect of status epilepticus, Hesdorffer [/bib_ref]. It is associated with high mortality (up to 20%), morbidity and considerable costs to the healthcare system [bib_ref] Status epilepticus in adults, Betjemann [/bib_ref] and can cause severe damage to the brain, leading to serious neurological complications such as cognitive impairment [bib_ref] Irreversible brain injury following status epilepticus, Korngut [/bib_ref] , and the development of chronic epilepsy [bib_ref] Risk of unprovoked seizure after acute symptomatic seizure: effect of status epilepticus, Hesdorffer [/bib_ref]. Where seizures are recurrent and spontaneous, epilepsy is diagnosed. According to the International League Against Epilepsy (ILAE), epilepsy is defined by any of the following conditions: "(1) at least two unprovoked (or reflex) seizures occurring > 24 h apart; (2) one unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years; and (3) diagnosis of an epilepsy syndrome" [bib_ref] ILAE official report: a practical clinical definition of epilepsy, Fisher [/bib_ref]. Epilepsy is one of the most common neurological disorders, globally. With an incidence of ∼1%, epilepsy affects over 65 million people worldwide [bib_ref] Epilepsy: new advances, Moshe [/bib_ref]. This is associated with a global disease burden of 7M disability adjusted life years (DALYs) [bib_ref] The global burden of epilepsy, Leonardi [/bib_ref] and with an estimated annual cost of over €20 billion in Europe alone according to the World Health Organization (2010). Beside the occurrence of spontaneous seizures, epilepsy is associated with an increased mortality and co-morbidities such as anxiety and depression, which severely impact quality of life [bib_ref] Epilepsy: new advances, Moshe [/bib_ref]. Epilepsy affects people of all ages, but is most common in the young and, particularly, the elderly [bib_ref] Incidence of epilepsy is now higher in elderly people than children, Everitt [/bib_ref]. Epilepsy can either be innate or acquired, arise due to genetic mutations or via epigenetic mechanisms [bib_ref] Genetic and epigenetic mechanisms of epilepsy: a review, Chen [/bib_ref] , structural or metabolic alterations [bib_ref] Limbic networks: clinical perspective, Reid [/bib_ref] , infection and immune dysregulation [bib_ref] The role of inflammation in epilepsy, Vezzani [/bib_ref] , some combination thereof or, as is often the case, be of unknown etiology (idiopathic epilepsy) [bib_ref] Idiopathic focal epilepsies: the "lost tribe, Pal [/bib_ref]. Common mutations underlying epilepsy include those affecting the function of ion channels, such as the Na + channel, Voltage-Gated Sodium Channel Alpha Subunit (SCN1A) [bib_ref] Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around..., Kasperaviciute [/bib_ref] , reducing the action potential threshold in neurons. Structural causes often arise as a result of changes in neuronal network connectivity following an initial insult to the brain, such as head injury, stroke, or status epilepticus [bib_ref] Epileptogenesis. Cold Spring Harb, Pitkanen [/bib_ref]. Epileptogenesis, the process of a normal brain becoming epileptic, is usually the result of a precipitating injury and characterized by an interplay of factors including ongoing cell death, inflammation and synaptic and axonal plasticity changes [bib_ref] Epileptogenesis. Cold Spring Harb, Pitkanen [/bib_ref]. Temporal lobe epilepsy (TLE), the most prevalent form of acquired epilepsy, is characterized by hippocampal sclerosis, including neuronal loss, mossy fiber sprouting and the formation of aberrant neuronal networks which can form a unilateral seizure focus, typically in the CA3 region of the hippocampus [bib_ref] Hippocampal neurodegeneration, spontaneous seizures, and mossy fiber sprouting in the F344 rat..., Rao [/bib_ref] , from which seizures often generalize. Possibly because of the importance of these network changes, TLE is associated with a particularly high prevalence of pharmacoresistance. ## Current treatments for epilepsy and status epilepticus Over 25 AEDs are currently used in the clinic [bib_ref] Key factors in the discovery and development of new antiepileptic drugs, Bialer [/bib_ref]. Despite the relatively large range of options available, where the mechanisms of action are understood, they fit into three broad categories: increasing inhibitory transmission (e.g., the glutamate decarboxylase catalyst, Gabapentin), decreasing excitatory transmission [e.g., the non-competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor antagonist, Perampanel] and blockade of voltage-gated ion channels (e.g., Na + channel blocker, lamotrigine) [bib_ref] Key factors in the discovery and development of new antiepileptic drugs, Bialer [/bib_ref]. In most cases, AEDs have multiple actions and are incompletely understood. For example, Topiramate exerts an inhibitory effect on Na + conductance, enhances GABA neurotransmission via unknown mechanisms, and antagonizes AMPA receptors [bib_ref] An overview of the preclinical aspects of topiramate: pharmacology, pharmacokinetics, and mechanism..., Shank [/bib_ref]. While there is a superficial diversity in mechanisms, all treatment options rely on the concept of redressing a balance between excitatory and inhibitory drive. This has proven largely successful in controlling seizures, but no treatments have been developed that act on the emergence or progression of the epileptic condition. Further, approximately 30% of patients remain pharmacoresistant to all available AEDs; in most cases leaving surgery as their sole remaining option [bib_ref] Epilepsy: new advances, Moshe [/bib_ref]. Choice of treatment strategy is based on seizure type, epilepsy syndrome, health problems, other medication used, lifestyle of the patients and considerations such as pregnancy [bib_ref] Epilepsy: new advances, Moshe [/bib_ref] [bib_ref] Epilepsy in pregnancy, Kinney [/bib_ref]. AEDs are the frontline treatment for epilepsy. Although strides have been made in terms of safety, tolerability, and pharmacokinetics with the new generation of AEDs, such as felbamate, gabapentin, lamotrigine or oxcarbazepine, the number of patients resistant to all treatments has not moved from 30% for approximately 80 years [bib_ref] Epilepsy: new advances, Moshe [/bib_ref] and the search for mechanisms which could disrupt the emergence or progression of the disease remains elusive. There is therefore an urgent need to identify new drug targets which can show efficacy in patients who are currently refractory to available treatment, and can demonstrate a disease modifying effect. When treating status epilepticus, time is a key factor and terminating the seizure is the number one priority for preventing lasting damage. A protocol for treatment of status epilepticus has been developed whereby, a first line treatment is administered within 5-10 min of seizure onset, a second line treatment is administered within 20-40 min and a third line treatment around 60 min following seizure onset [bib_ref] The drug treatment of status epilepticus in Europe: consensus document from a..., Shorvon [/bib_ref]. The best first line treatment is with benzodiazepines, such as lorazepam, diazepam, or midazolam [bib_ref] Status epilepticus in adults, Betjemann [/bib_ref]. Evidence supporting the best treatment strategy for second and third line treatments is weaker, however current practice involves the use of AEDs such as fosphenytoin, valproic acid or levetiracetam [bib_ref] Evidence-based guideline: treatment of convulsive status epilepticus in children and adults: report..., Glauser [/bib_ref] and anesthetic drugs [bib_ref] Status epilepticus in adults, Betjemann [/bib_ref]. As with epilepsy, approximately 30% of status epilepticus patients are refractory to available drug treatment and these patients are particularly vulnerable to adverse clinical outcomes [bib_ref] Refractory status epilepticus: a prospective observational study, Novy [/bib_ref]. In summary, the drug development challenges for epilepsy and status epilepticus are similar, with a need in both cases for drugs which show efficacy in currently pharmacoresistant patients, while reducing comorbidities and adverse drug effects. In the case of epilepsy, preventing the emergence or progression of the disorder is also an important goal. ## New directions in drug development for epilepsy While drugs targeting excitatory and inhibitory drive have proven widely successful in controlling seizures, it seems likely that in order to modify disease progression or offer efficacious drug treatment to currently pharmacoresistant epilepsy patients, alternative targets, with a novel mechanism of action, must be sought. Several experimental and clinical findings have demonstrated an important role for neuroinflammation in both icto-and epileptogenesis [bib_ref] The role of inflammation in epilepsy, Vezzani [/bib_ref] [bib_ref] Infections, inflammation and epilepsy, Vezzani [/bib_ref]. High levels of inflammatory mediators are present in the brains of both experimental rodent models of epilepsy and epilepsy patients [bib_ref] Neuroinflammatory targets and treatments for epilepsy validated in experimental models, Aronica [/bib_ref] and these processes have therefore received much attention in recent years. Selective blockade of the pro-inflammatory cytokine, Interleukin-1β (IL-1β), has been shown to reduce seizures in in vivo models of epilepsy [bib_ref] Interleukin converting enzyme inhibition impairs kindling epileptogenesis in rats by blocking astrocytic..., Ravizza [/bib_ref] [bib_ref] Basic mechanisms of status epilepticus due to infection and inflammation, Vezzani [/bib_ref] , while in an epileptogenesis-resistant animal, the Amazon rodent, Proechimys, no acute brain inflammatory response was found following experimentally-induced status epilepticus [bib_ref] Status epilepticus does not induce acute brain inflammatory response in the Amazon..., Scorza [/bib_ref]. Following an insult, such as a seizure or period of status epilepticus, pro-inflammatory cytokines, such as IL-1β, tumor necrosis factor-α (TNF-α) and IL-6 are released in the brain, primarily from astrocytes and microglia [bib_ref] Inflammation and Epilepsy: preclinical findings and potential clinical translation, Terrone [/bib_ref]. These pro-inflammatory cytokines exert a number of effects that contribute to a reduction in the seizure threshold and emergence of chronic epilepsy. Experimental evidence demonstrates that pro-inflammatory cytokines can have an effect on the firing properties of neurons directly, through the modulation of voltage-gated Na + , Ca 2+ , and K + ion channels [bib_ref] Cytokines and neuronal ion channels in health and disease, Viviani [/bib_ref] , facilitation of excitatory neurotransmission through both pre-and post-synaptic mechanisms, and disinhibition via antagonism of GABA A receptors [bib_ref] The stress-induced cytokine interleukin-6 decreases the inhibition/excitation ratio in the rat temporal..., Garcia-Oscos [/bib_ref]. The effect of inflammation on seizures and epilepsy, however, is not limited to direct modulation of the excitatory/inhibitory balance. Gliosis, gliotransmission, increased permeability of the bloodbrain barrier (BBB) and subsequent influx of peripheral cells and modulatory molecules, neuronal cell death and the aberrant reorganization of neuronal networks can all be consequences of a neuroinflammatory response [bib_ref] Inflammation and epilepsy, Vezzani [/bib_ref]. The causality between hyperexcitation, excitotoxicity, and neuroinflammation is circular and, as described below, intercellular signaling through purines is an important mediator of these processes, making purinergic receptors an attractive treatment target. ## Purinergic signaling It was not until 1972 that the role of adenosine-5 -triphosphate (ATP) as an intercellular molecule, was first described by . Today, it is well-recognized that a wide variety of nucleotides, including ATP, function as either sole or co-transmitter in both the peripheral and central nervous system (CNS). ATP can act as a fast, excitatory neurotransmitter or as a neuromodulator and is involved in a vast array of short-and long-term physiological and pathological processes including inflammation, cellular survival, proliferation, cellular differentiation, and synaptic plasticity [bib_ref] Purinergic signalling: from normal behaviour to pathological brain function, Burnstock [/bib_ref] [bib_ref] Neuromodulation by extracellular ATP and P2X receptors in the CNS, Khakh [/bib_ref] [bib_ref] Nucleotide signalling during inflammation, Idzko [/bib_ref]. It has therefore been implicated in numerous different diseases of the CNS including epilepsy [bib_ref] Purinergic signalling: therapeutic developments, Burnstock [/bib_ref]. ## Purine release in the brain Purines and pyrimidines are a well-established source of energy in all living cells. These molecules, however, also play an important role in intercellular communications within the CNS [bib_ref] Uracil nucleotides: from metabolic intermediates to neuroprotection and neuroinflammation, Lecca [/bib_ref] [bib_ref] Nucleotide signalling during inflammation, Idzko [/bib_ref]. Adenine and uridine nucleotides are present in almost every synaptic and secretory vesicle where they are either present alone, functioning as a fast neurotransmitter or co-stored with classical neurotransmitters (e.g., GABA or glutamate) [bib_ref] Purinergic signalling in the nervous system: an overview, Abbracchio [/bib_ref]. Under physiological conditions, adenine and uridine nucleotides are usually present at micromolar concentrations in the extracellular space; however, under pathological conditions (e.g., inflammation, hyperexcitability, and cell death) extracellular nucleotide levels can reach the milimolar range [bib_ref] Release of adenosine and ATP during ischemia and epilepsy, Dale [/bib_ref] [bib_ref] Nucleotide signalling during inflammation, Idzko [/bib_ref] [bib_ref] ATP as a multi-target danger signal in the brain, Rodrigues [/bib_ref]. ATP [and most likely uridine-5 -triphosphate (UTP)] can enter the extracellular space by crossing the compromised membranes of damaged and dying cells [bib_ref] ATP as a multi-target danger signal in the brain, Rodrigues [/bib_ref]. In addition, purines are actively released from different cell types including neurons, astrocytes, microglia, and endothelial cells to act as neuro-and glio-transmitters [bib_ref] Uracil nucleotides: from metabolic intermediates to neuroprotection and neuroinflammation, Lecca [/bib_ref] [bib_ref] ATP as a multi-target danger signal in the brain, Rodrigues [/bib_ref]. Several mechanisms have been proposed to contribute to the release of nucleotides into the extracellular medium including cell damage, exocytosis of secretory granules, vesicular transport involving the vesicular nucleotide transporter (VNUT) and membrane channels such as ABC transporters, pannexins, connexins and via purinergic receptors themselves [bib_ref] Uracil nucleotides: from metabolic intermediates to neuroprotection and neuroinflammation, Lecca [/bib_ref] [bib_ref] ATP as a multi-target danger signal in the brain, Rodrigues [/bib_ref]. Once released into the extracellular space, adenine and uridine nucleotides are rapidly metabolized by ectonucleotidases (e.g., ectonucleoside triphosphate diphosphohydrolases, ectonucleotide pyrophosphatase, alkaline phosphatases, ecto-5 -nucleotidase, and ecto-nucleoside diphosphokinase) into different breakdown products including adenosine-5 -diphosphate (ADP), adenosine, uridine-5 -diphosphate (UDP), and uridine. These metabolites, in turn, are important neurotransmitters/neuromodulators in their own right, with specific receptors for each expressed throughout the CNS [bib_ref] Ectonucleotidases in the nervous system, Zimmermann [/bib_ref] [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref]. Direct evidence for ATP release during seizures is mixed. Large elevations in ATP on electrical stimulation of the cortex [bib_ref] Distribution and release of adenosine triphosphate in rat brain, Wu [/bib_ref] provided the first direct evidence that high levels of neuronal activity could induce the release of ATP. Subsequently, stimulation of the Schaffer collateral in hippocampal slices was demonstrated to induce ATP release in a Ca 2+ -dependent, but glutamate receptor activation-independent manner , suggesting the release of ATP was pre-synaptic. While ATP release was not detected following high frequency stimulation or electrically-induced epileptiform seizure like events in hippocampal slices [bib_ref] Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations, Lopatar [/bib_ref] , the induction of epileptiform activity in rat hippocampal slices with the use of the mGluR5-agonist, (S)-3,5-Dihydroxyphenylglycine induced the release of ATP through pannexin hemichannels [bib_ref] Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations, Lopatar [/bib_ref]. ATP release was also elevated in hippocampal slices in a high K + model of seizures [bib_ref] K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat..., Heinrich [/bib_ref]. [bib_ref] Variations of ATP and its metabolites in the hippocampus of rats subjected..., Dona [/bib_ref] used microdialysis and high-performance liquid chromatography in order to attempt to measure extracellular concentrations of ATP and its metabolites in vivo after pilocarpine-induced status epilepticus and following the onset of chronic epilepsy. They found no change in ATP concentrations for 4 h following status epilepticus, but a marked increase in ATP metabolites, including adenosine monophosphate (AMP) and ADP. Concentrations of ATP and all metabolites were reduced during chronic epilepsy, but ATP was elevated by 300% during spontaneous seizures. Because ectonucleotidases rapidly hydrolyze ATP in the extracellular space and the concentration and activity of these enzymes are increased following seizures [bib_ref] GABA synapses and the rapid loss of inhibition to dentate gyrus granule..., Naylor [/bib_ref] , it is difficult to measure changes in ATP release directly. Less interest has been shown in investigating UTP release following seizures, however, [bib_ref] UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis, Koizumi [/bib_ref] demonstrated that following kainic acid (KA)-induced-seizure-like events in hippocampal slices, extracellular concentrations of UTP were elevated approximately threefold [bib_ref] UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis, Koizumi [/bib_ref]. Whereas the anticonvulsive properties of the nucleoside, adenosine, are well-documented [bib_ref] Adenosinergic signaling in epilepsy, Boison [/bib_ref] , the possible contribution of extracellular nucleotides to seizure pathology is a relatively new research area . The discovery of increased extracellular levels of ATP in seizure-prone rats was one of the first studies to suggest a functional contribution of extracellular nucleotides to seizures . Demonstrating a direct impact on seizures, another early study showed that the microinjection of ATP analogs into the prepiriform cortex led to the generation of motor seizures [bib_ref] Adenosine and ATP in epilepsy, Knutsen [/bib_ref]. More recent evidence implicating extracellular nucleotides in seizure generation stems from studies showing that the injection of ATP into the brain of mice led to the development of high spiking on the electroencephalogram (EEG) and exacerbated seizure severity during status epilepticus [bib_ref] Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status..., Engel [/bib_ref] [bib_ref] Neurodevelopmental alterations and seizures developed by mouse model of infantile hypophosphatasia are..., Sebastian-Serrano [/bib_ref]. In contrast, treatment with UTP decreases the rate of neuronal firing in epileptic rats [bib_ref] Uridine modulates neuronal activity and inhibits spike-wave discharges of absence epileptic Long..., Kovacs [/bib_ref] and in mice subjected to status epilepticus [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. Further, UTP metabolites such as uridine reduce epileptic seizures in patients with epileptic encephalopathy [bib_ref] CAD mutations and uridine-responsive epileptic encephalopathy, Koch [/bib_ref]. ## P2 receptor family Once released, extracellular adenine and uridine nucleotides bind to and activate specific cell surface receptors termed P2 receptors which are ubiquitously expressed and functional on all cell types in the CNS [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref]. The P2 family of receptors include the ionotropic P2X channels and the metabotropic P2Y receptors. The fast acting P2X channels are a family of seven cation-permeable ionotropic receptor subunits (P2X1-7) which form both homo-and hetero-trimers, depolarizing the cell membrane upon activation [bib_ref] P2X receptors as cell-surface ATP sensors in health and disease, Khakh [/bib_ref]. All P2X receptors are activated by their main endogenous agonist, ATP, and are permeable to small cations including Na + , K + , and Ca 2+ . All P2X receptor subunits share a common topology with two transmembrane domains, a large extracellular loop and an intracellular amino and carboxyl terminus [bib_ref] P2X receptors as cell-surface ATP sensors in health and disease, Khakh [/bib_ref] [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref]. Much attention has been paid to the study of P2X receptors over the past decades, in particular in diseases of the CNS [bib_ref] Purinergic signalling: from normal behaviour to pathological brain function, Burnstock [/bib_ref] [bib_ref] Modulation of the neuronal network activity by P2X receptors and their involvement..., Saez-Orellana [/bib_ref]. P2X receptor activation has been implicated in numerous pathological conditions including neurodegeneration, inflammation, ischemia, brain trauma, and hyperexcitability [bib_ref] Purinergic signalling: therapeutic developments, Burnstock [/bib_ref]. Among the P2X receptor subtypes, the P2X7 receptor has attracted by far the most attention as a potential therapeutic target for brain diseases [bib_ref] P2X7 receptor: an emerging target in central nervous system diseases, Sperlagh [/bib_ref] [bib_ref] The evolution of P2X7 antagonists with a focus on CNS indications, Rech [/bib_ref]. While the P2X receptor family is made up of fast acting ligand-gated ion channels, the metabotropic P2Y receptor family consists of eight G-protein coupled slower-acting receptors: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 [bib_ref] Pharmacological profiles of cloned mammalian P2Y-receptor subtypes, Von Kugelgen [/bib_ref] [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref]. In contrast to P2X channels, P2Y receptors can be activated by more than one substrate including the adenine nucleotides ATP (P2Y 2 and P2Y 11 ) and ADP (P2Y 1 , P2Y 12 , and P2Y 13 ) and the uridine nucleotides UTP (P2Y 2 and P2Y 4 ), UDP (P2Y 6 and P2Y 14 ), and UDPglucose (P2Y 14 ). P2Y receptors contain the typical features of G-protein-coupled receptors which includes an extracellular amino terminus, intracellular carboxyl terminus and seven transmembrane-spanning motifs [bib_ref] Nucleotides acting at P2Y receptors: connecting structure and function, Jacobson [/bib_ref]. P2Y receptors can be further subdivided into groups based on their coupling to specific G proteins. P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors are coupled to Gq proteins, which stimulate phospholipase C, ultimately resulting in the subsequent release of Ca 2+ from intracellular stores and activation of protein kinase C (PKC). Of these, P2Y 11 receptor can also couple to Gs, stimulating adenylate cyclase and increasing the production of cyclic adenosine monophosphate (cAMP) [bib_ref] Pharmacological profiles of cloned mammalian P2Y-receptor subtypes, Von Kugelgen [/bib_ref]. P2Y 12 , P2Y 13 , and P2Y 14 are coupled to Gi proteins, inhibiting adenylate cyclase and thereby decreasing cAMP production [bib_ref] Pharmacological profiles of cloned mammalian P2Y-receptor subtypes, Von Kugelgen [/bib_ref]. ## Involvement of p2y receptor signaling in brain inflammation and excitability P2Y receptors are involved in a myriad of different cellular functions and pathological processes pertinent to the process of epileptogenesis and epilepsy including neuroinflammation, neurodegeneration, synaptic reorganization, and changes in neurotransmitter release [bib_ref] Pharmacological profiles of cloned mammalian P2Y-receptor subtypes, Von Kugelgen [/bib_ref] [bib_ref] P2Y nucleotide receptors: promise of therapeutic applications, Jacobson [/bib_ref] [bib_ref] Epileptogenesis. Cold Spring Harb, Pitkanen [/bib_ref] [bib_ref] P2Y receptors in synaptic transmission and plasticity: therapeutic potential in cognitive dysfunction, Guzman [/bib_ref] making them an attractive antiepileptic therapeutic target. Inflammatory processes in the brain have received much attention over recent years and are thought to play a major role in seizure-induced pathology and the development of epilepsy [bib_ref] The role of inflammation in epilepsy, Vezzani [/bib_ref]. The principle ligands for P2Y receptors are the purine, ATP, the pyrimidine, UTP, and their metabolites, such as ADP and UDP [bib_ref] Physiology and pathophysiology of purinergic neurotransmission, Burnstock [/bib_ref]. The role of each receptor in neuroinflammation is dictated by its affinity for different ligands and downstream targets. ATP is both released as a result of inflammation and promotes proinflammatory mechanisms. This circular causality can underpin a positive feedback loop whereby neuroinflammation becomes self-sustaining [bib_ref] Nucleotide signalling during inflammation, Idzko [/bib_ref]. Less is known about the role of UTP in mediating neuroinflammation. The role of different P2Y receptors in mediating neuroinflammation and cell death seems to be divergent [bib_ref] Supportive or detrimental roles of P2Y receptors in brain pathology?-The two faces..., Forster [/bib_ref] , depending on downstream signaling pathways and mutually antagonistic actions, but is incompletely understood. The P2Y 1 receptor, activated by the ATP metabolite ADP, is expressed also on astrocytes and activated under conditions of oxidative stress, prompting the release of IL-6 [bib_ref] P2Y1 receptor signaling enhances neuroprotection by astrocytes against oxidative stress via IL-6..., Fujita [/bib_ref]. IL-6 has been shown to play an anti-inflammatory role during 'classic signaling' involving the binding of IL-6 to the membranebound IL-6 receptor which induces the dimerization of the β-receptor glycoprotein 130 (gp130). In contrast however, IL-6 is also critical for pro-inflammatory signaling in a process termed 'trans-signaling, ' whereby IL-6 stimulates distant cells which only express gp130 in the absence of the IL-6 receptor [bib_ref] The role of interleukin-6 signaling in nervous tissue, Rothaug [/bib_ref]. A more recent study has shown that in a chronic model of epilepsy, astrocytes from kindled rats show enhanced Ca 2+ -dependent signaling and astroglial hyperexcitability, which requires the activation of the P2Y 1 receptor [bib_ref] Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain, Alvarez-Ferradas [/bib_ref]. P2Y 1 antagonism prevented cognitive deficits and neuronal damage in a model of ischemia in mice. A recent study also showed improved histological and cognitive outcomes in a model of TBI in mice provided by P2Y 1 receptor antagonism [bib_ref] Antagonism of purinergic signalling improves recovery from traumatic brain injury, Choo [/bib_ref]. Activation of astrocytic P2Y 2 receptors promotes astrocyte activation and migration via an interaction with αV-integrin [bib_ref] Molecular basis of self-sustaining seizures and pharmacoresistance during status epilepticus: the receptor..., Wang [/bib_ref]. The P2Y 2 receptor has also been shown to play a protective role against chronic inflammation-induced neurodegeneration in a model of Alzheimer's disease . A role for the uridine-sensitive P2Y 4 receptor in mediating neuroinflammation has not been established [bib_ref] Purinergic mechanisms in neuroinflammation: an update from molecules to behavior, Beamer [/bib_ref] , with progress hamstrung by a lack of specific tools for targeting this receptor. The P2Y 6 receptor promotes the activation of microglia and the adoption of a phagocytic phenotype following activation by the UTP metabolite UDP [bib_ref] UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis, Koizumi [/bib_ref]. This is dependent on downstream signaling involving phospholipase C and PKC. Other studies have suggested a role for the P2Y 12 receptor in microglial activation [bib_ref] P2Y12 receptor-mediated integrin-beta1 activation regulates microglial process extension induced by ATP, Ohsawa [/bib_ref] , showing that activation of integrin-β1 in microglia through P2Y 12 is involved in directional process extension by microglia in brain tissue. As discussed in more detail below, P2Y 12 -dependent process extension has been shown to be increased following status epilepticus in mice [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref]. The effects of P2Y signaling are not limited to inflammatory processes and cellular survival alone. P2Y signaling also impacts directly on neuronal excitability, synaptic strength, and synaptic plasticity [bib_ref] P2Y receptors in synaptic transmission and plasticity: therapeutic potential in cognitive dysfunction, Guzman [/bib_ref]. Presynaptic P2Y receptors have been shown to affect the release of different neurotransmitters including glutamate, noradrenaline and GABA, most likely by reducing presynaptic Ca 2+ influx [bib_ref] Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured..., Fischer [/bib_ref]. P2Y 1 , P2Y 2 , and P2Y 4 inhibit the release of glutamate in the hippocampus [bib_ref] ATP inhibits glutamate synaptic release by acting at P2Y receptors in pyramidal..., Mendoza-Fernandez [/bib_ref] [bib_ref] Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures, Koizumi [/bib_ref] [bib_ref] Dual presynaptic control by ATP of glutamate release via facilitatory P2X1, P2X2/3,..., Rodrigues [/bib_ref] , possibly through the inhibition of voltage-activated Ca 2+ channels (VACCs) [bib_ref] Inhibition of N-type voltage-activated calcium channels in rat dorsal root ganglion neurons..., Gerevich [/bib_ref]. Using the same mechanism, the release of noradrenaline in the hippocampus was also blocked via P2Y 1 , P2Y 12 , and P2Y 13 activation. Similarly, activation of P2Y 4 with UTP blocks the release of the inhibitory neurotransmitter GABA from cerebellar basket cells [bib_ref] GABA release by basket cells onto Purkinje cells, in rat cerebellar slices,..., Donato [/bib_ref]. P2Y receptors alter the expression/function of other membrane receptors and voltage-gated ion channels. P2Y 1 triggers the desensitization or internalization of the metabotropic glutamate receptor 1 (mGluR1) [bib_ref] Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous..., Mundell [/bib_ref] and inhibits N-methyl-D-aspartate (NMDA) receptor channels [bib_ref] P2Y(1) receptor activation inhibits NMDA receptor-channels in layer V pyramidal neurons of..., Luthardt [/bib_ref]. P2Y 1 also increases the sensitivity of the GABA A receptor [bib_ref] Metabotropic P2Y purinoceptor-mediated presynaptic and postsynaptic enhancement of cerebellar GABAergic transmission, Saitow [/bib_ref] and inhibits P2X receptors [bib_ref] Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their..., Gerevich [/bib_ref]. P2Y receptor activation can lead to the inhibition of VACCs (Diverse-Pierluissi et al., 1991) thereby potentially influencing neuronal excitability and synaptic plasticity. P2Y receptors also block potassium channels [e.g., voltage-gated potassium channel subunit KvLQT2,3 [bib_ref] Activation of P2Y1 nucleotide receptors induces inhibition of the M-type K+ current..., Filippov [/bib_ref] or G protein-coupled inward rectifying channels 1, 2, and 4 (GIRK1,2,&4)] [bib_ref] Activation and inhibition of neuronal G proteingated inwardly rectifying K(+) channels by..., Filippov [/bib_ref] , inhibiting membrane hyperpolarisation and thereby facilitating an increased frequency of neuronal firing [bib_ref] Neural KCNQ (Kv7) channels, Brown [/bib_ref] [bib_ref] P2Y receptors in synaptic transmission and plasticity: therapeutic potential in cognitive dysfunction, Guzman [/bib_ref]. On a network level, P2Y 1 increases the firing of GABAergic inhibitory neurons either directly or via P2Y 1 -dependent activation of astrocytes in the hippocampus, eventually leading to an increase in inhibitory-postsynaptic currents (IPSCs) in pyramidal neurons [bib_ref] ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks, Bowser [/bib_ref]. In a more recent study, [bib_ref] P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat..., Jacob [/bib_ref] showed that astrocytic P2Y 1 activation increases extracellular concentrations of GABA by inhibiting Ca 2+ signaling dependent GABA transport [bib_ref] P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat..., Jacob [/bib_ref]. In conclusion, while P2X receptors excerpt a mainly facilitatory effect on synaptic transmission [bib_ref] Neuromodulation by extracellular ATP and P2X receptors in the CNS, Khakh [/bib_ref] , the effects of P2Y receptors seem to be context-specific, either increasing or decreasing neuronal firing by altering excitatory and inhibitory neurotransmitter release or altering receptor function (e.g., NMDA and GABA A ) and channel conductance (e.g., voltage-gated KCNQ2/3 potassium channel) [bib_ref] P2Y receptors in synaptic transmission and plasticity: therapeutic potential in cognitive dysfunction, Guzman [/bib_ref]. ## Purinergic signaling as a novel drug target in epilepsy Mounting evidence has accumulated over the past decades demonstrating a causal role for purinergic signaling in numerous pathological conditions ranging from cancer (Di [bib_ref] Purines, purinergic receptors, and cancer, Virgilio [/bib_ref] , cardiovascular disease [bib_ref] P2X receptors in the cardiovascular system and their potential as therapeutic targets..., Ralevic [/bib_ref] , blood cell diseases [bib_ref] Neural regulation of inflammation in the airways and lungs, Mcgovern [/bib_ref] to diabetes [bib_ref] P2X receptors and diabetes, Fotino [/bib_ref] and brain diseases [bib_ref] P2X and P2Y receptors-role in the pathophysiology of the nervous system, Puchalowicz [/bib_ref]. Among brain diseases, intervention in purinergic signaling has been postulated as a new therapeutic avenue for acute insults to the brain such as stroke [bib_ref] Targeting P(2)X(7) receptor for the treatment of central post-stroke pain in a..., Kuan [/bib_ref] and TBI [bib_ref] Activation of P2X7 promotes cerebral edema and neurological injury after traumatic brain..., Kimbler [/bib_ref] and for chronic brain diseases including neurodegenerative diseases (e.g., Huntington's, Alzheimer's, and Parkinson's disease) [bib_ref] Nucleotides in neuroregeneration and neuroprotection, Miras-Portugal [/bib_ref] , neuropsychiatric disorders (e.g., depression and schizophrenia) [bib_ref] Purinergic signalling: from normal behaviour to pathological brain function, Burnstock [/bib_ref] and also epilepsy [bib_ref] The ATP-gated P2X7 receptor as a target for the treatment of drug-resistant..., Beamer [/bib_ref]. Emphasizing the potential for targeting purinergic signaling as a promising new therapeutic strategy, several compounds are already used in the clinic, including the P2Y 2 agonist Diquafosol for the treatment of dry eye [bib_ref] P2Y2 receptor agonists for the treatment of dry eye disease: a review, Lau [/bib_ref] or Clopidogrel, a P2Y 12 antagonist used for the treatment of thrombosis [bib_ref] Clinical use of clopidogrel, Sarafoff [/bib_ref] while others have progressed into clinical trials such as antagonists of the ionotropic P2X3 used against refractory chronic cough [bib_ref] P2X3 receptor antagonist (AF-219) in refractory chronic cough: a randomised, double-blind, placebo-controlled..., Abdulqawi [/bib_ref] and P2X7 receptors used against rheumatoid arthritis [bib_ref] Clinical evaluation of the efficacy of the P2X7 purinergic receptor antagonist AZD9056..., Keystone [/bib_ref] and other inflammatory conditions [bib_ref] The evolution of P2X7 antagonists with a focus on CNS indications, Rech [/bib_ref]. To date, most of the studies performed to elucidate the changes in expression and functional contribution of purinergic P2 receptors to seizures and epilepsy have focused on the P2X receptor subtype, in particular the P2X7 receptor (reviewed in [bib_ref] The ATP-gated P2X7 receptor as a target for the treatment of drug-resistant..., Beamer [/bib_ref] , with relatively little attention paid to the P2Y receptor family. The lack of apparent interest was largely due to a lack of suitable tools (e.g., drugs to manipulate P2Y function) and the strong focus on fast synaptic effects conferred by the ionotropic P2X receptors . Recent studies using experimental animal models of status epilepticus and epilepsy and analysis of patient brain tissue, however, suggest a prominent role for P2Y signaling during seizures and the development of epilepsy [fig_ref] TABLE 1 |: Summary of the main findings of P2Y receptor expression and function during... [/fig_ref]. In the last section of this review we describe in detail the evidence linking a pathological activation of the metabotropic P2Y receptors to seizure generation and seizureinduced pathology and discuss the antiepileptic potential of drugs targeting P2Y signaling. ## P2y expression following status epilepticus One of the earliest studies analyzing P2Y expression changes following status epilepticus used the intraperitoneal KA-induced status epilepticus mouse model [bib_ref] Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic..., Avignone [/bib_ref]. Here, the authors observed an increase in transcription of P2ry 6 , P2ry 12 , and P2ry 13 in the hippocampus. In another study using the intraperitoneal pilocarpine mouse model,show an increase in P2Y 1 activity in neuronal progenitor cells following status epilepticus. In a more recent study, our group published a comprehensive analysis of changes in transcription and expression across the entire P2Y family of receptors following status epilepticus using two different mouse models: the intraamygdala KA mouse model of status epilepticus [bib_ref] Unilateral hippocampal CA3-predominant damage and short latency epileptogenesis after intra-amygdala microinjection of..., Mouri [/bib_ref] and the intraperitoneal pilocarpine mouse model of status epilepticus [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. Both, intraamygdala KA and intraperitoneal pilocarpine-induced status epilepticus increased the transcription of the uridine-sensitive P2Y receptors P2ry 2 , P2ry 4 , and P2ry 6 in the hippocampus. At the same time, the transcription of the adenine-sensitive receptors P2ry 1 , P2ry 12 , and P2ry 13 was downregulated. At the protein level, hippocampal levels of P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 were increased and P2Y 12 was decreased following status epilepticus. No immunohistochemistry was performed to identify cell types expressing the different P2Y receptors. Thus, these results show that changes in the transcription of P2Y receptors following status epilepticus closely correlate with the known profile of agonists (i.e., adenine-sensitive receptors are downregulated and uridine-sensitive receptors are upregulated) and, at the protein level, the G-protein coupling of the receptors with P2Y receptors coupled to Gq being increased and P2Y receptors coupled to Gi being downregulated or not changed [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. ## P2y expression during chronic epilepsy Much less is known about the expression profile of P2Y receptors during epilepsy. To date, the only study carried out characterizing P2Y expression in experimental epilepsy was undertaken using the intraamygdala KA mouse model [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. In this model, mice become epileptic after a short latent period of 2-5 days [bib_ref] Unilateral hippocampal CA3-predominant damage and short latency epileptogenesis after intra-amygdala microinjection of..., Mouri [/bib_ref]. Analysis of the hippocampus 14 days-post status epilepticus revealed increased P2ry 1 , P2ry 2 , and P2ry 6 transcription and increased P2Y 1 , P2Y 2 , and P2Y 12 protein levels. No changes were observed for the remaining receptors. Thus, P2Y upregulation seems to be the predominant response during experimental epilepsy, probably due to an increase in inflammatory processes in the epileptic brain. In the same study, resected hippocampal samples from drug-refractory epilepsy patients were also analyzed. In these samples, as seen before in hippocampal samples from epileptic mice, the predominant response was an upregulation of P2Y receptors with P2Y 1 and P2Y 2 significantly upregulated. Of note, the only exception, and in contrast to findings from the mouse model of epilepsy, expression of the P2Y 13 receptor was found at lower levels in the epileptic brain compared to controls [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. In another previous study using brain tissue from patients suffering from intractable epilepsy associated with focal cortical dysplasia, [bib_ref] Expression of astrocyte-related receptors in cortical dysplasia with intractable epilepsy, Sukigara [/bib_ref] showed increased levels of P2Y 1 , P2Y 2 , and P2Y 4 . Interestingly, the authors reported the main increase to be in astrocytes [bib_ref] Expression of astrocyte-related receptors in cortical dysplasia with intractable epilepsy, Sukigara [/bib_ref]. Thus, P2Y receptor expression is altered during epilepsy, however, in contrast to status epilepticus, the main response was an upregulation of the P2Y receptor family. ## P2y function during status epilepticus Despite the involvement of P2Y signaling in numerous pathological processes believed to play a key role during epilepsy, a possible involvement of the different P2Y receptor subtypes to seizure-induced pathology remains poorly explored and only three recent studies have suggested a functional contribution of P2Y receptors to seizures or seizure-induced pathology. The first study demonstrating a causal role for P2Y signaling during status epilepticus used mice deficient in P2Y 12 [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref]. P2Y 12 is one of the most important therapeutic targets of the P2Y receptor family, with P2Y 12 agonists already routinely used in the clinic as an antithrombotic agent [bib_ref] P2Y12 receptors: structure and function, Cattaneo [/bib_ref]. [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref] report a P2Y 12 -dependent extension of microglial process toward neurons following KA-induced status epilepticus. Neuronal NMDA receptor activation led to an influx of Ca 2+ , stimulating ATP release, which subsequently activated microglial P2Y 12 receptors, which, in turn stimulated the extension of the processes. Interestingly, P2Y 12 knockout mice, in which this process was inhibited, showed an increased seizure severity [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref]. Thus, the authors concluded that microglial P2Y 12 receptors are necessary for microglia-neuron interaction during status epilepticus and that microglial process extension via P2Y 12 may serve an anti-ictal function. In a later study, [bib_ref] Altered morphological dynamics of activated microglia after induction of status epilepticus, Avignone [/bib_ref] demonstrate that microglial processes extend toward a pipette containing methylthio-ADP, an agonist for P2Y 1 , P2Y 12 , and P2Y 13 (and a weak agonist for P2Y 11 ). The velocity of this chemotaxis was increased in activated microglia following status epilepticus. Because they also found an upregulation of P2Y 12 in activated microglia, the authors attributed this receptor as the likely mediator of this response [bib_ref] Altered morphological dynamics of activated microglia after induction of status epilepticus, Avignone [/bib_ref]. More recently, our group has shown seizure altering properties of the broad-spectrum P2Y agonists ADP and UTP in the intraamygdala KA mouse model [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. Once status epilepticus was established, mice treated with ADP showed an increased seizure severity and mice treated with UTP showed a strong reduction in seizure severity and accompanying seizureinduced cell death [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. These results are in line with protective cellular mechanisms acting during status During and following status epilepticus, purines (e.g., ATP and UTP) are actively released from different cell types including neurons, astrocytes and microglia or enter the extracellular space from damaged/necrotic cells. While the activation of UTP/UDP-sensitive P2Y receptors (P2Y 2 , P2Y 4 , and P2Y 6 ) may has anticonvulsive and neuroprotective effects, the activation of ATP/ADP-sensitive P2Y receptors (P2Y 1 and P2Y 13 ) may be pro-convulsive. The role of P2Y 11 and P2Y 14 have not been studied so far and therefore are unknown. Counterintuitively, P2Y 12 is ATP/ADP-sensitive, but seems to be anticonvulsive and neuroprotective. epilepticus regarding the P2Y receptor family with adeninesensitive receptors being generally downregulated during status epilepticus and uridine-sensitive receptors being upregulated [fig_ref] FIGURE 1 |: Divergent effects on seizures and seizure-induced pathology of different P2Y agonists [/fig_ref]. In conclusion, while these results demonstrate a causal role for P2Y signaling during status epilepticus, we are still far from a clear and comprehensive picture of how individual P2Y receptors impact on seizure pathology. ## P2y function during chronic epilepsy Although results from functional studies during status epilepticus and changes in expression of P2Y receptors during epilepsy strongly suggest a role for these receptors in epilepsy, to date, no studies have been performed to determine the functional contribution of P2Y receptors to epileptogenesis or the epileptic phenotype. Possible reasons are the lack of centrally available P2Y-targeting drugs and the lack of mouse models with conditional deletion of P2Y receptors, both essential for the study of the involvement of P2Y receptors during epilepsy. ## Conclusion and future perspectives What remains to be done to establish P2Y receptors as potential drug target for epilepsy in the future? Despite the exciting emerging data revealing P2Y signaling in the brain, we are only at the beginning of understanding the potential role in seizure generation and during epileptogenesis. Recent studies have shown distinct changes in expression of the P2Y receptor family following status epilepticus and during seizures and a functional contribution has been postulated using broad-spectrum P2Y agonists (ADP and UTP) [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref] and P2Y 12 knockout mice [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref] , there are many key issues, however, which will have to be resolved before considering P2Y receptors as valid drug target. (i) Studies have demonstrated altered P2Y receptor expression following status epilepticus and during epilepsy [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. To get a better picture about the potential role of P2Y signaling during seizure-related pathologies, however, we must determine what cell types (e.g., neurons vs. glia; inhibitory vs. excitatory neurons) express the receptor and their sub-cellular localization (e.g., somatic vs. synaptic). (ii) Treatment of mice during status epilepticus with P2Y broad-spectrum agonists suggest a role of these receptors in seizure generation and seizure-induced pathology [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref] , however, we still do not know the role of individual P2Y receptors during seizures, with the only exception being the P2Y 12 receptor [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref]. P2Y receptor-specific, centrally available drugs or P2Y knock-out mice, if possible cell-specific, must be used to determine the possible impact of the different P2Y receptors on seizures and epilepsy. (iii) P2Y receptors have been shown to be involved in numerous pathological processes in the brain [bib_ref] Purinergic mechanisms in neuroinflammation: an update from molecules to behavior, Beamer [/bib_ref] , however, signaling downstream of P2Y during seizures and epilepsy remains elusive, with the only exception being P2Y 12 functioning on microglia [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref]. P2Y receptors have been shown to alter both excitatory (e.g., glutamate) and inhibitory neurotransmitter release in the brain [bib_ref] The stress-induced cytokine interleukin-6 decreases the inhibition/excitation ratio in the rat temporal..., Garcia-Oscos [/bib_ref] , therefore, future studies must determine whether P2Y signaling impacts on the release of neurotransmitters and what neurotransmitters are altered during seizures. Do seizure-induced changes in P2Y function impact on the function of other cell membrane channels/receptors (e.g., potassium channels, calcium channels, NMDA receptors, GABA receptors) thereby altering neuronal excitability? (iv) Different P2Y receptors respond to different agonists (e.g., UTP, UDP, ATP, and ADP) [bib_ref] Pharmacological profiles of cloned mammalian P2Y-receptor subtypes, Von Kugelgen [/bib_ref] , however, we still do not know at what concentrations these nucleotides are available during seizures/epilepsy and when, where and from which cell types these nucleotides are released or what mechanisms (e.g., ectonucleotidases) are responsible for extracellular nucleotide concentration changes. (v) To date, studies have solely used the KA and pilocarpine mouse model of status epilepticus to analyze P2Y signaling during seizures [bib_ref] Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic..., Avignone [/bib_ref] [bib_ref] Altered morphological dynamics of activated microglia after induction of status epilepticus, Avignone [/bib_ref] [bib_ref] Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12..., Eyo [/bib_ref] [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref]. These mouse models rely, however, on chemically-induced seizures and only recapitulate certain aspects of the disease [bib_ref] Experimental models of status epilepticus and neuronal injury for evaluation of therapeutic..., Reddy [/bib_ref]. Results must therefore be confirmed in other models of acute seizures and chronic epilepsy. (vi) To date, we do not know what drives P2Y receptor expression during seizures. The clear expression pattern according to P2Y receptor agonists during status epilepticus, however, points toward common pathways. The identification of what drives P2Y expression during and following seizures may also therefore provide much needed new target genes for seizure control. (vii) While changes in P2Y receptor expression and, to an extent, function, have been analyzed in hippocampal tissue, extrahippocampal brain areas, in particular the cortex, may also contribute to the epilepsy phenotype [bib_ref] Cognitive decline in severe intractable epilepsy, Thompson [/bib_ref] [bib_ref] Cognitive outcome of status epilepticus in adults, Helmstaedter [/bib_ref]. Status epilepticus is associated with significant extrahippocampal injury, including in the cortex [bib_ref] Status epilepticus-induced neuronal loss in humans without systemic complications or epilepsy, Fujikawa [/bib_ref] and cortical thinning has also been reported in patients with pharmacoresistant TLE [bib_ref] Cortical thickness analysis in temporal lobe epilepsy: reproducibility and relation to outcome, Bernhardt [/bib_ref]. Consequently, the P2Y expression profile must also be analyzed in non-hippocampal brain regions. (viii) Data obtained by using the broad-spectrum agonists ADP and UTP with ADP exacerbating and UTP decreasing seizure pathology [bib_ref] Expression and function of the metabotropic purinergic P2Y receptor family in experimental..., Alves [/bib_ref] , suggest that a mix of antagonist (e.g., adeninespecific receptors) and agonists (e.g., uridine-specific receptors) may provide better protection than single receptor targeting. In conclusion, P2Y signaling is altered during and after status epilepticus and during epilepsy. Functional studies demonstrate an involvement of P2Y receptors in seizure pathology. Despite promising results, however, we are only at the beginning of understanding the role of P2Y signaling during seizures to ultimately establish P2Y targeting as possible therapeutic avenue in epilepsy. [fig] FIGURE 1 |: Divergent effects on seizures and seizure-induced pathology of different P2Y agonists. (A) Representative EEG traces recorded during intraamygdala KA-induced status epilepticus showing pro-convulsive effects of ADP treatment, while UTP acts as potent anti-convulsive. (B) UTP protects against neuronal cell death following status epilepticus, visualized with the neuronal-cell death marker Fluoro-Jade B. (C) Hypothetical model of P2Y receptor function during and after status epilepticus or damaging seizures. [/fig] [table] TABLE 1 |: Summary of the main findings of P2Y receptor expression and function during status epilepticus and epilepsy in experimental models of epilepsy and patient brain. [/table]

Bedside EEG predicts longitudinal behavioural changes in disorders of consciousness A B S T R A C TProviding an accurate prognosis for prolonged disorder of consciousness (pDOC) patients remains a clinical challenge. Large cross-sectional studies have demonstrated the diagnostic and prognostic value of functional brain networks measured using high-density electroencephalography (hdEEG). Nonetheless, the prognostic value of these neural measures has yet to be assessed by longitudinal follow-up. We address this gap by assessing the utility of hdEEG to prognosticate long-term behavioural outcome, employing longitudinal data collected from a cohort of patients assessed systematically with resting hdEEG and the Coma Recovery Scale-Revised (CRS-R) at the bedside over a period of two years. We used canonical correlation analysis to relate clinical (including CRS-R scores combined with demographic variables) and hdEEG variables to each other. This analysis revealed that the patient's age, and the hdEEG theta band power and alpha band connectivity, contributed most significantly to the relationship between hdEEG and clinical variables. Further, we found that hdEEG measures recorded at the time of assessment augmented clinical measures in predicting CRS-R scores at the next assessment. Moreover, the rate of hdEEG change not only predicted later changes in CRS-R scores, but also outperformed clinical measures in terms of prognostic power. Together, these findings suggest that improvements in functional brain networks precede changes in behavioural awareness in pDOC. We demonstrate here that bedside hdEEG assessments conducted at specialist nursing homes are feasible, have clinical utility, and can complement clinical knowledge and systematic behavioural assessments to inform prognosis and care. Recently, we aimed to address this need by conducting a longitudinal assessment of 39 pDOC patients to describe the long-term trajectory of behavioural awareness(Bareham et al., 2019). Patients were assessed at the bedside with the Coma Recovery Scale-Revised (CRS-R) once every 3 months for a period of 2 years, allowing for the assessment of longer-term outcomes. The analysis showed statistically significant recovery in behaviour recorded by the CRS-R, with improvements observed beyond 12 months post injury -supporting the proposal to abandon diagnoses of permanence(Giacino et al., 2018). # Introduction ## The prognostic challenge in pdoc Clinicians face a difficult challenge at predicting the longer-term outcome of pDOC patients following brain injury. This is in part due to a lack of studies that have tracked patients systematically to characterise the history of recovery. Longitudinal studies that undertake systematic follow-up of such patients are challenging as, after acute care, many patients are transferred to specialist neurological centres, nursing homes or repatriated to the family home, with incomplete records of the clinical course and outcomes. As such, most of the studies of pDOC are cross-sectional with convenience samples of patients that do not inform the journey of recovery. Clinical practice guidelines highlight the need for more longitudinal studies to assist with understanding long-term recovery following severe brain injury . This finding highlighted that many patients may show evidence of late behavioural improvements, a pattern in the natural history of recovery that can be detected with systematic longitudinal follow-up. Also in line with the update to United States (U.S.) practice guidelines, the study identified that patients' age, time since injury and initial CRS-R diagnosis were important predictors of long-term recovery, highlighting these factors as important for prognostication. The study also formally assessed, for the first time, the effect of arousal on CRS-R trajectories. Arousal had a significant effect on the behavioural assessment of awareness and was a significant predictor of behavioural trajectories. ## Neuroimaging methods to assist prognostication Recent advances in neuroimaging have indicated that certain neural correlates are associated with the state of consciousness. In particular, research with functional Magnetic Resonance Imaging (fMRI) has indicated that default mode network activity is associated with conscious state in pDOC. fMRI has also been the prominent approach to detect covert awareness in a minority of patients. Problematically, fMRI is unlikely to be viable as a neuroimaging tool for diagnosis and prognosis as it is often not available, feasible for the patient, or affordable. Without regular follow ups to capture variable and delayed changes in behavioural awareness, the prognostic value of fMRI assessments for pDOC is difficult to determine. High density electroencephalography (hdEEG) is a technique that can be used for regular and repeated assessment at the patient's bedside. Resting state functional brain networks measured with hdEEG have been shown to be associated with behavioural state in pDOC. In particular, structured networks of alpha band connectivity have been shown to reflect the level of behavioural awareness in both patientsand in healthy people transitioning in and out of sedation. This hdEEG indicator of consciousness has also been shown to have prognostic value. This research motivated the prospective BETADOC (Bedside Test of Awareness in Disorders of Conscious) study, to demonstrate the prognostic utility of this hdEEG measure by longitudinally monitoring individual patients at the bedside to determine whether accurate estimates of brain network activity can predict behavioural changes. Using case studies from the BETADOC study, we have already demonstrated that hdEEG measures can capture the stability and recovery of behavioural awareness over time. Moreover, the hdEEG measures shown to best discriminate between groups of patientswere similarly shown to track the progression across conscious states within the context of an individual patient. These promising findings suggest that hdEEG could have clinical value for prognostication. ## The betadoc study With a novel framework that employs a combination of behavioural and brain-based methods to assess patients' consciousness state, here we report findings from one of the first longitudinal multimodal studies that systematically follows up a group of pDOC patients. Patients were assessed at the bedside using both the CRS-R and resting state hdEEG measures once every 3 months for a period of 2 years. In total, we collected and analysed 185 assessments from 40 patients recruited across the course of the study. Firstly, we conducted Canonical Correlation Analysis (CCA) to relate the hdEEG and clinical measures to each other. This approach allows for the analysis of the association between two sets of data -the hdEEG measures that capture the structure of functional brain networks on the one hand, and clinical (including behavioural and demographic) measures on the other. Previous analysis of these clinical measures identified arousal as an important predictor of behavioural recovery. Given that previous research has identified that these hdEEG measures are closely linked with consciousness state, we predicted that arousal will similarly be an important correlate of the longitudinal trajectory of the hdEEG measures. Secondly, we used the canonical scores from the CCA, which independently summarise the pattern of hdEEG and clinical data over time, to determine whether these measures can be used to prognosticate behavioural changes in awareness. # Materials and methods ## Standard protocol approvals, registrations, and patient consents This study was carried out in accordance with the United Kingdom (UK) National Health Service Research Ethics Committee for Cambridgeshire (reference: 16/EE/0006) recommendations. Patients' consultee, or in the absence of a suitable consultee the ward manager acted as a nominated consultee, provided written informed consent prior to enrolment in accordance with the UK Mental Capacity Act 2005 and Declaration of Helsinki. ## Patients description Patients were recruited from and assessed at two specialist neurological rehabilitation centres, where they received consistent and specialised care throughout the study. For study inclusion, patients needed to be aged 16 years or older and clinically diagnosed as pDOC following any form of sudden onset, non-progressive brain injury. They had to be referred to, or under review of, a consultant in rehabilitation medicine or consultant neurologist. Patients were excluded in the instance of pregnancy, if they were clinically unstable or, if they were diagnosed with a serious mental health condition prior to their brain injury that has required active management by a psychiatrist. Patients who emerged from a DOC immediately prior to recruitment were also excluded. We retained patients who emerged from pDOC during the study, although no further assessments were conducted on these patients after their emergence (see . All medications were recorded at the time of recruitment. Forty patients were recruited, however one patient died prior to the first scheduled assessment. The analyses presented here are based on the remaining 39 patients (see. Of these, 16 had an initial CRS-R diagnosis of UWS, 15 were MCS-(Minimally conscious minus; no evidence of command following) and 7 MCS+ (Minimally conscious plus; evidence of command following) and 1 EMCS (Emerged from a minimally conscious state). MCS-and MCS + patients were categorised based on the level of complexity of observed behaviours, consistent with the definition from Bruno et al.. 18 patients had an aetiology of traumatic brain injury (TBI), with the remaining 22 had an anoxic (14), stroke (5) or other (2). Over the course of the 2-year data collection period, 24 patients changed CRS-R diagnosis; 14 progressed from UWS to MCS-/MCS+/EMCS (N = 1) and 10 from MCS-/+ to MCS+/EMCS. The patient that initially had a CRS-R diagnosis of EMCS declined on later assessments to an MCS + diagnosis (see supplementary . A subset of data from patients 3, 10, 18 and 21 were reported in a previous study. ## Design The same researcher (CAB) assessed the patients once every 3 months at the bedside using the CRS-R to determine changes in behaviour as well as hdEEG at rest (see. The CRS-R was conducted prior to the hdEEG recording to ensure hdEEG was recorded at patient's peak arousal. # Data availability statement In total, data from 183 assessments from 39 patients were included in the analysis and has been made available in the supplementary material. ## Coma recovery scale-revised The CRS-R is a 23-item scale behavioural assessment of awareness for pDOC. The scale is split into auditory, visual, motor, oromotor/verbal, communication, and arousal subscales. Where possible, the patient was assessed upright in the chair. If this was not possible, patients were assessed at the bedside with the bed elevated to an upright sitting position. When it was required, the arousal intervention of applying deep pressure as per the CRS-R guidelines was administered prior to and, if necessary, throughout the duration of the examination to ensure the patient maintained peak possible arousal. ## High-density eeg resting state Fifteen minutes of resting state data was collected using a 128channel saline electrode net (Phillips Neuro/Electric Geodesics Inc.) Data were collected at a sampling rate of 500 Hz and were later downsampled to 250 Hz offline. Prior to EEG collection, the CRS-R was administered to assist with ensuring patients were awake with their eyes open. Patients' behaviours and EEG data were monitored online to ensure recordings were free from seizure activity. The EEG pre-processing and artifact rejection method was identical to(see supplementary methods). An average of 15 electrodes were rejected and interpolated (range 1-43). The number of rejected trials range from 1 to 53 (Mean = 17). ICA was used to remove muscle artefacts, eyeblinks and in some occasions heartbeat artefacts. On average, 30 such noisy ICA components were removed (range 2-53). The data analysis pipeline was identical(see supplementary methods). Briefly, the debiased weighted phase lag index (dwPLI)metric of connectivity was estimated and graph theory metrics were computed to describe the pattern of this connectivity between electrodes. These measures and the 3D network topography (seewere visualised alongside those from controls and other patient groups in a feedback reports given to family, in addition to being entered to statistical models. The data analysis pipeline was implemented using EEGLAB, Fieldtrip , the Brain Connectivity Toolbox, and custom MATLAB scripts. The pipeline was automated except for manual checks for and removal of artifactual channels, trials and independent components. # Canonical correlation analysis Canonical correlation analysis (CCA) seeks maximal correlations between two sets of data. Drawing on the approach used by, we were interested in the correlation between the hdEEG variables on the one hand, and the clinical variables on the other. The 13 clinical variables were Age, Aetiology, Days since onset of injury, Gender, Initial Diagnosis (CRS-R diagnosis at the first assessment), Patient Number and Assessment Number, as well as each of the scores on the CRS-R subscales. The hdEEG variables included the mean and standard deviation of Patients are ordered by time of recruitment into the study, and those recruited later had fewer assessments at the end of the 2-year study period. C. Data Processing Pipeline for Connectivity Analysis -Methodology was identical to. Cross-spectral density between pairs of channels was estimated using dwPLI. Resulting connectivity matrices were proportionally thresholded. Thresholded connectivity matrices were visualized as topographs, which combined information about the topography of connectivity with the modular topology of the network. Graph-theoretic metrics were then calculated after binarising the thresholded connectivity matrices. the spectral power, as well as the median and standard deviation (over the 91 electrodes) of the dwPLI connectivity in the delta, theta and alpha frequency bands. Additionally, we computed graph-theoretic variables summarising the connectivity "network", by thresholding, binarising and then modelling the 91 × 91 connectivity matrix as graphs. These graph measures were estimated at each connection density threshold between 0.1 and 0.9, in steps of 0.025. Briefly, the clustering coefficient of a network captures its local efficiency, while the characteristic path length measures the average topological distance between pairs of nodes in a graph, providing a measure of global efficiency. Modularity, calculated here using the Louvain algorithm, is a network metric that captures the degree to which the nodes of a network can be parcellated into densely connected, topologically distinct modules. Given a modular decomposition, the participation coefficient of a node is an inter-modular measure of its centrality. Finally, modular span is average weighted topographical distance (over the scalp) spanned by a module identified in a network. To combine the graph theory values across multiple thresholds before entering them into the CCA, Principal component analysis (PCA) was run over band-wise values at computed thresholds, and the score of the first component was included, which explained an average of 72% (SD = 8.7%) of the variance. In total, we included 42 variables derived from hdEEG, 14 in each of the 3 frequency bands (see for a full list of hdEEG variables). The clinical (N = 13) and EEG (N = 42) datasets were submitted to CCA using the canoncorr function in MATLAB. The CCA was run with significance testing estimated non-parametrically, using 2000 randomised permutations of the rows of the behavioural and demographic variables relative to the hdEEG variables. These permutations, while destroying the statistical relationship between the variables, respected the repeated-measures structure of the data and shuffled the order of the patients while preserving all the assessments from each patient in the same relative order. The resulting CCA produces a set of canonical variates that captures the linear combination of variables that produces the strongest correlation between the two datasets. Each variate has a score for each patient's assessment (183 scores per variate). ## Linear mixed effects model To investigative the potential additive prognostic value of the hdEEG to the clinical measures, we fitted a Linear Mixed Effects Model (LMEM). Values from the EEG and clinical canonical variates (calculated by the CCA above) from the previous assessment were entered as predictors of the current CRS-R score. As described above, the canonical variate is effectively a weighted mixture of one group of variables that is maximally correlated with a weighted mixture of another group of variables. We entered the canonical variate in the LMEM, instead of the EEG measures themselves, to reduce the dimensionality of the model and thereby increase sensitivity of the LMEM analysis. Only patients with at least two clinical and hdEEG assessments conducted at two separate time points could be included in this analysis (N = 36 patients, 144 data points). The model included a random factor of patient number to account for the different number of observations per patient. The model also included the intercept of time since the first assessment (assessment number) to account for the longitudinal nature of the data. The dependent variable of CRS-R score was normalised using a Gaussian rank inverse normalisation methodto achieve a Gaussian distribution of values and avoid any influence of potential outliers. The predictors of clinical and hdEEG canonical variate (CV) scores from the previous assessment were also normalised to avoid any influence of outliers. The analysis was conducted using syntax in SPSS software, where the LMEM model equation was as follows: = + + y intercept(assessment number) random factor (patient number) fixed factors(clinical variate, hdEEG variate) ## Linear stepwise regression To understand how the rate of change in the hdEEG measures might predict future change in CRS-R scores, we conducted a linear stepwise regression on the data from patients that had at least 4 assessments (N = 23 patients, 46 difference scores). The choice of 4 completed assessments was motivated by the fact that including patients with 5 or more assessments reduced the number of patients and statistical power to a degree that did not allow for formal analysis. We took the rate of change (Δ) of both the hdEEG and the clinical CV values from Assessments 1 to 2 and entered these as predictors of the change in normalised CRS-R scores from Assessments 3 to 4. Using a backward stepwise methodology, predictors were removed sequentially to generate the final model that best explained future CRS-R changes. # Results ## Correlating hdeeg with clinical progression CCA was used to investigate the relationship between the EEG and clinical measures. The first three pairs of canonical variates (CV) were significantly correlated. However, the first canonical correlation r = 0.77, p = < 0.001 was the strongest and explained the most variance R= 0.033 (see . Pearson's correlations indicated that age was the best predictor of the EEG CV, followed by Arousal (See . Notably, age was significantly negatively associated with the EEG canonical scores indicating that high EEG scores were more likely associated with younger patients. Arousal on the other hand was significantly positively correlated indicating that higher EEG scores was associated with higher arousal scores on the CRS-R. These findings are in line with the U.S. clinical practice guidelinesand recent findings that age and arousal subscores were important predictors of CRS-R outcome. The strongest correlations between the EEG variables and the first clinical CV were negative correlations with mean and standard deviations of theta power and a positive correlation with clustering and mean dwPLI connectivity in alpha (see . This indicates that reductions in theta power and increases in alpha connectivity best predicted changes in CRS-R scores over time. This is in line withthat demonstrated that dwPLI participation coefficient in the alpha band tracked improvements in CRS-R for a patient that progressed from a UWS to MCS-CRS-R diagnosis. ## Predicting clinical progression using hdeeg To understand whether hdEEG had any additive value over and above the clinical measures at predicting future CRS-R score, we fitted a Linear Mixed Effects Model (LMEM) to the normalised CRS-R scores predicted by the clinical and hdEEG CVs from the previous assessment. This approach enabled us to enter our data into a single statistical correlation model, without having to resort to pairwise comparisons and consequent correction for multiple comparisons. The LMEM indicated that the assessment number (time) and the EEG CV from the previous assessment were significant predictors of CRS-R score (see , Model 1). However, the clinical CV from the previous assessment was not a significant predictor. We removed this non-significant predictor and refitted the model (see , Model 2) which improved the fit, evidenced by the reduced −2 log likelihood (see indicating the reduced amount of variance left . The Linear Mixed Effects Models of the CRS-R score predicted by the scores on the EEG and clinical canonical variates from the previous assessment using backwards stepwise methodology. Non-significant predictors are removed one by one from the model. The shaded model was the winner, in which a patient's current CRS-R was predicted only by the previous value of their hdEEG canonical variate. A bar chart of the −2 Log likelihood values is presented for each model. The yellow shaded model 2 had the lowest log likelihood, indicating that it had the relatively lowest amount of unexplained variance. B. The Linear Mixed Effects Models of the patient's current CRS-R score predicted by the hdEEG canonical variate and CRS-R score from the previous assessment using backwards stepwise methodology. The winning model only included the patient's previous CRS-R score to predict the current one. A bar chart is presented of the −2 Log likelihood values for each model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) unexplained. For completeness, we fitted Model 3 to evaluate the predictive power of the clinical CV alone on future CRS-R scores. The increased −2 Log likelihood confirmed (see that removal of the hdEEG CV reduced predictive power and led to a poorer model fit. To determine whether the poor predictive power of the clinical CV was due to its composition of demographic variables with CRS-R subscores, we refitted the LMEM, but this time replacing the clinical CV with normalised CRS-R scores from the previous assessment instead (See . With this model, the hdEEG CV did not explain any variance over and above the CRS-R score from the previous assessment. In this case, the best-fitting model (Model 2) included only the Assessment Number and the previous CRS-R score, confirmed by comparison of the −2 Log likelihoods (see , with the variance left unexplained reduced by removal of the hdEEG CV. ## Predicting future changes in crs-r A limitation of the modelling above was that predicting the subsequent CRS-R score with the previous CRS-R score may lead to an improved model fit from identical scaling. As such, the hdEEG CV may be a poorer predictor simply because the EEG metrics vary on a different scale. To address this, we conducted a subsequent analysis to investigate the prognostic value of the rate of change (Δ) of the hdEEG CV and CRS-R scores on future CRS-R changes. By calculating the rate of change, we put the EEG and normalised CRS-R onto a similar scale, allowing for a fairer juxtaposition of EEG and clinical measures. Moreover, this approach investigated whether early changes in hdEEG predicts later changes in CRS-R scores.presents the results of the backwards stepwise linear regression. The final winning model removed the CRS-R (note the nonsignificant t-value of -.856p = 0.402) and instead selected Δ hdEEG CV from Assessment 1 to 2 as the best predictor of Δ CRS-R from Assessment 3 to 4. The predictive power of this winning model is reflected with the increased amount of variance explained (Adjusted R 2 ) in comparison to the model including the Δ Normalised CRS-R score predictor (see. Finally, we found that the Δ hdEEG CV was also a better predictor than Δ clinical CV (see; note the t value of −2.982, p = 0.007 for the Δ hdEEG CV whilst Δ clinical CV had a t of only 0.933 and nonsignificant p value = 0.362). The linear fit of the Δ hdEEG CV to the Δ future CRS-R is visualised in. Whilst the figure demonstrates a negative correlation between the Δ hdEEG CV and Δ future CRS-R, likely driven by reductions in theta power (the strongest variable that correlated with the hdEEG CV), the strength of the correlation is the most important feature. # Discussion The BETADOC study has taken a longitudinal approach to assess pDOC patients at the bedside in their resident neurological centre using both behavioural and brain-based methods. This novel framework has allowed for regular, systematic follow-ups with pDOC patients to assess the value of both hdEEG and the CRS-R at predicting long-term outcomes. Our analysis has indicated that the hdEEG measures of theta power and the connectivity measures of clustering and median dwPLI in the alpha band are the strongest contributors to the canonical scores of clinical-behavioural trajectories. Also, patients' age and arousal levels, already identified in our previous analysisas. A. Linear Stepwise regression of the change in a patient's CRS-R scores from assessment 3 to 4 predicted by the change in their hdEEG canonical variate and normalised CRS-R scores in assessments 1 to 2. Models are ordered using backwards stepwise methodology with non-significant predictors removed sequentially. The winning model, shaded grey, included only the hdEEG variates as predictors. A bar chart of adjusted R2 values for each model is presented, with the winning model with largest R 2 highlighted in yellow. B. Linear stepwise regression of the change in CRS-R scores from assessment 3 to 4 predicted by the change in hdEEG and clinical canonical variates from assessment 1 to 2. Comparison of this model to the winning model in panel A shows that this model has less predictive power than the model that includes only the hdEEG canonical variate alone. C. Figure illustrating the linear relationship between the change in CRS-R scores from assessment 3 to 4 and the change in hdEEG canonical variates from assessment 1 to 2, captured by the winning model in panel A. Each grey circle is a single patient (N = 23). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) important predictors of behavioural recovery, were also the strongest correlates of hdEEG canonical variates. In addition, our findings show that hdEEG measures, when combined with clinical measures, improved our ability to predict consequent CRS-R scores. Moreover, our findings show that the rate of hdEEG changes early in the trajectory is the best predictor of later behavioural changes, outperforming the predictive power of early behavioural changes. These findings indicate that resting state hdEEG measures have significant prognostic value for predicting long-term behavioural outcomes in pDOC. ## The link between brain and behaviour in pdoc recovery We took a statistically valid approach (CCA) to assess the relationship between patterns of hdEEG and clinical measures over time. This enabled us to understand how brain and behaviour related to each other in this complex and variable clinical cohort. In doing so, this study extends beyond most of previous research that has pinpointed neural measures most strongly associated with consciousness state usually measured once in a patient, using a cross-sectional design. Here, over 183 assessments collected systematically from 39 patients every 3 months, we have identified that reductions in theta power and increases in alpha clustering and connectivity are the most important hdEEG measures that correlate with patients' clinical profiles. This finding is consistent with cross-sectional studies showing that reduced theta powerand increases in alpha band connectivity is associated with consciousness state across patient groupsand with recovery of individual cases. Therefore, reduced theta band activity and increased alpha band network connectivity are indicators of higher consciousness levels, or recovery, in individual patients over time. This finding has important clinical implications for the development of a brain-based tool for diagnosis and prognosis. Targeting theta power and alpha connectivity could improve the sensitivity and accuracy of methods that aim to measure consciousness and the potential for recovery. The CCA approach also allowed us to assess the clinical measures most strongly related with hdEEG network changes over time. We found that along with patients' age, arousal was an important correlate of hdEEG. We have previously reported that age and arousal are important correlates of behavioural recovery. While the role of arousal is clinically known to be important, this is the first time that arousal has been formally tested as an important correlate of brain-based measures of consciousness. ## Arousal is an important predictor of hdeeg trajectories Alongside the behavioural assessment of consciousness, an intervention may be required to ensure peak arousal before conducting neuroimaging assessments or when using Brain Computer Interfaces. Also, the influence of arousal is an important consideration in the development of neuroimaging methods for diagnosis and prognosis in this patient cohort. Research into computational methods that aim to produce a valid neural index of consciousness need to appropriately account for fluctuations in arousal that mediate other neural correlates of consciousness such as connectivity and complexity. Consciousness involves an interaction between two theoretical concepts of arousal and awareness. Whilst arousal is thought to capture the level of consciousness, awareness is considered to capture conscious content. Our findings demonstrated that changes in arousal correlated with changes in hdEEG measures over time. This finding agrees with the notion that emergence from DOC involves recovery of arousal systems that support brain networks that underpin conscious awareness, perhaps revealing the course of neural mechanisms in the natural history of recovery following brain injury. This proposal is in line with the meso-circuit hypothesis, and could explain the beneficial effects of interventions such as amantadine and zolpidem, and deep brain stimulation, that target regions of the brainstem and thalamus involved in arousal modulation leading to increases in large scale cortical projections that support conscious awareness. ## The value of bedside hdeeg to prognosticate behavioural recovery We directly assessed the prognostic value of the hdEEG measures combined with some of the clinical measures typically used in current settings to assist with clinical decisions of prognosis and care plans. The central finding from our LMEM was that the addition of hdEEG to these clinical measures improved our ability to predict the CRS-R score in the next assessment. This finding implicates that hdEEG adds significant value to prognostication when combined with clinical measures. The current CRS-R score itself was of course a very good predictor of the CRS-R score on a subsequent assessment. But this is not entirely surprising given that, in this case, the independent (predictor) variable and dependent variable are measured on the same scale, whereas the hdEEG canonical scores have undergone a scale transformation. To determine whether hdEEG is a valuable inclusion to patient assessments we investigated whether early hdEEG changes predict later changes in CRS-R scores. This analysis avoided the scaling issue by keeping all the variables on comparable scales. Our results indicated that not only did change in the hdEEG CV predict later CRS-R changes, hdEEG outperformed the prognostic ability of earlier changes in CRS-R scores. The implication of this finding is that hdEEG would not only be a valuable contribution to routine clinical care to track changes in awareness and assist with diagnosis, it would arm clinicians with stronger evidence to provide accurate prognoses and inform rehabilitation and care plans. More generally, this finding also indicates that changes in brain network measures captured with hdEEG precede changes in behaviour. This finding has been alluded to in previous cross-sectional studies using hdEEGbut not formally assessed in a longitudinal study until now. Information regarding changes in brain network connectivity may provide more fine-grained information regarding the potential for recovery that presages the behavioural manifestation of consciousness. As such, hdEEG assessments are likely to be more valuable than behavioural measures of awareness to predict behavioural outcomes. ## Clinical utility of bedside hdeeg The current UK clinical practice guidelines note that resting state EEG was not considered to have discriminative utility to assist with diagnosis in pDOC (Physicians RCo, 2020). Since these guidelines have been published, EEG measures of resting state networks have been found to capture important information that can discriminate between diagnostic groups, and these measures have been shown to be robust enough to significantly predict outcome in pDOC. Here, we have utilised these resting state hdEEG measures to systematically assess patients at the bedside in their resident nursing homean approach that did not require relocation of patients to a specialised setting. This approach highlights the feasibility of potentially conducting bedside EEG assessments regularly as part of routine care to allow for long-term systematic follow up and improved detection of late changes in awareness. We showed a strong correlation between hdEEG and clinical measures of pDOC over time implicating that hdEEG can provide corroborative evidence for improved pDOC diagnosis. This could be particularly useful when behavioural measures are inconsistent due to arousal fluctuations. Future studies could assess whether sensitivity of hdEEG to arousal fluctuations and whether hdEEG variability can predict CRS-R variability, potentially assisting diagnosis in complex cases. Moreover, arousal was found to be a significant predictor of both behavioural trajectoriesand hdEEG trajectories in these patients. It is possible that fine grained, rich, hdEEG measures of arousal may surpass the CRS-R arousal subscore in terms of prognostic power. This notion is substantiated by the finding that the rate of hdEEG change surpassed changes in CRS-R in terms of predicting later behavioural changes. As such, hdEEG can improve prognostic information to assist clinical decisions and direct treatment care plans and allocation of therapeutic resources. From a practical perspective, high-density EEG saline net takes approximately 10 min to prepare, on par with low-density EEG systems using conventional gel electrodes. Hence, hdEEG systems could be feasible for clinical bedside assessments despite the density of electrodes. # Limitations Research has indicated that a minimum of 5 CRS-R assessments are required to ensure consistency . In this study, patients were assessed only once every 3 months as multiple assessments were not feasible as the scale of this study only allowed for a single examiner. The arousal sub-scale has been included as a variable in the study to account for potential fluctuations and the examiner administered the arousal intervention when considered necessary, as per CRS-R guidelines, to ensure peak arousal prior to assessment. Nevertheless, the use of a single examiner could have led to undetected measurement bias. Future larger scale studies should employ multiple examiners to establish inter-rate reliability between examiners on longitudinal assessments. Here, we have used the gaussian normalised CRS-R score as an indicator of behavioural change. However, increases in CRS-R do not necessarily indicate improved consciousness state. This is accounted for in the CCA with CRS-R diagnosis included as a variable in the analysis, but this was not able to be included in LMEM analyses. Nonetheless, the LMEM results still captures whether EEG can predict behavioural changes as measured by CRS-R. Due to attrition, and because patients were recruited at any time up until 3 months before the end of data collection, there are a different number of assessments for each patient. To control for this potential source of variation, we included Patient Number as a random factor in statistical models. The patients reported here all resided in specialist centres with access to rehabilitation services that other patients may not have access to. To better characterise the role of the rehabilitation context on patient trajectories, further larger studies could involve patients from multiple centres. # Conclusions The strength of the longitudinal design in the BETADOC study has revealed important predictors of longer-term outcomes in pDOC. Using CCA, we identified the hdEEG measures most strongly linked with behavioural and demographic variables. The prognostic value of the hdEEG measures was evident when combined with clinical measures. Moreover, the utility of early hdEEG changes surpassed early behavioural changes in prognosticating later changes in behaviour. The addition of regular and repeated hdEEG to routine care then, will not just benefit current clinical assessments by providing corroborative evidence. Our findings here indicate that hdEEG assessments can further improve the accuracy of clinical prognostication. Crucially, hdEEG measures captured patients' potential for recovery with changes in hdEEG preceding behavioural changes. Finally, we note that arousal was also an important correlate of hdEEG measures and, as such, arousal levels should be taken into account during diagnostic and prognostic neuroimaging in pDOC. # Financial disclosures

Health and Economic Burden of Post-Partum Staphylococcus aureus Breast Abscess Objectives: To determine the health and economic burdens of post-partum Staphylococcus aureus breast abscess.Study design: We conducted a matched cohort study (N = 216) in a population of pregnant women (N = 32,770) who delivered at our center during the study period from 10/1/03-9/30/10. Data were extracted from hospital databases, or via chart review if unavailable electronically. We compared cases of S. aureus breast abscess to controls matched by delivery date to compare health services utilization and mean attributable medical costs in 2012 United States dollars using Medicare and hospital-based estimates. We also evaluated whether resource utilization and health care costs differed between cases with methicillin-resistant and -susceptible S. aureus isolates.Results: Fifty-four cases of culture-confirmed post-partum S. aureus breast abscess were identified. Breastfeeding cessation (41%), milk fistula (11.1%) and hospital readmission (50%) occurred frequently among case patients. Breast abscess case patients had high rates of health services utilization compared to controls, including high rates of imaging and drainage procedures. The mean attributable cost of post-partum S. aureus breast abscess ranged from $2,340-$4,012, depending on the methods and data sources used. Mean attributable costs were not significantly higher among methicillin-resistant vs.susceptible S. aureus cases.Conclusions: Post-partum S. aureus breast abscess is associated with worse health and economic outcomes for women and their infants, including high rates of breastfeeding cessation. Future study is needed to determine the optimal treatment and prevention of these infections. # Introduction Mastitis, most often caused by Staphylococcus aureus [bib_ref] Postdischarge surveillance following cesarean section: the incidence of surgical site infection and..., Del Monte [/bib_ref] [bib_ref] Prevalence of and risk factors for methicillin-resistant Staphylococcus aureus colonization and infection..., Maraqa [/bib_ref] [bib_ref] Wound infection after caesarean section, Moir-Bussy [/bib_ref] , occurs in approximately 20% of breastfeeding mothers and may be a precursor to the development of breast abscess [bib_ref] A descriptive study of mastitis in Australian breastfeeding women: incidence and determinants, Amir [/bib_ref] [bib_ref] Occurrence of lactational mastitis and medical management: a prospective cohort study in..., Scott [/bib_ref] [bib_ref] Incidence of mastitis in breastfeeding women during the six months after delivery:..., Kinlay [/bib_ref] [bib_ref] Mastitis in the first year postpartum, Vogel [/bib_ref]. S. aureus is also the predominant pathogen in post-partum breast abscess [bib_ref] Breast abscess bacteriologic features in the era of community-acquired methicillinresistant Staphylococcus aureus..., Moazzez [/bib_ref] [bib_ref] Community-acquired methicillin-resistant Staphylococcus aureus among patients with puerperal mastitis requiring hospitalization, Stafford [/bib_ref] [bib_ref] Risk factors for Staphylococcus aureus postpartum breast abscess, Branch-Elliman [/bib_ref] , and rates of methicillin-resistant S. aureus (MRSA) have increased in the pregnant and post-partum population [bib_ref] Risk factors for Staphylococcus aureus postpartum breast abscess, Branch-Elliman [/bib_ref] [bib_ref] Postpartum mastitis and community-acquired methicillin-resistant Staphylococcus aureus, Reddy [/bib_ref] [bib_ref] Incidence of methicillin-resistant Staphylococcus aureus in postpartum breast abscesses, Berens [/bib_ref]. Although breast abscess is a serious, uncommon complication of mastitis with high morbidity [bib_ref] MRSA breast abscesses in postpartum women, Chuwa [/bib_ref] [bib_ref] Methicillin-resistant Staphylococcus aureus infections may not impede the success of ultrasound-guided drainage..., Chen [/bib_ref] , health services utilized by patients with these infections have been poorly characterized. Neither the clinical outcomes, such as milk fistula formation and rates of breastfeeding cessation, nor the attributable medical costs of these infections are known. Further, it is unknown if the emergence of MRSA has worsened outcomes and increased health services utilization and medical cost. The aims of this study were to: (1) characterize the health and economic outcomes of patients with post-partum S. aureus breast abscess, such as breastfeeding cessation, development of milk fistulae, rates of adverse reactions to antibiotics, rates of health service utilization and medical costs and (2) explore whether costs are higher for MRSA vs. methicillin-susceptible S. aureus (MSSA) infections. # Methods ## Setting We conducted a population-based matched cohort study among post-partum women who delivered at Beth Israel Deaconess Medical Center (BIDMC), an academic tertiary care center with approximately 4,750 deliveries per year, between 10/1/2003-8/ 31/2010. From 10/2008-3/2010, a cluster of pulse-field type USA300-0114 MRSA infections in post-partum women and their infants who delivered at our center were identified; the majority of infections in mothers were mastitis and breast abscesses [bib_ref] Risk factors for Staphylococcus aureus postpartum breast abscess, Branch-Elliman [/bib_ref] [bib_ref] Medicare and Medicaid programs: hospital outpatient prospective payment; ambulatory surgical center payment;..., Wise [/bib_ref]. Cluster cases were defined as onset of MRSA post-partum breast abscess within one year after delivery. ## Case and control selection Cases were defined as any woman with a culture-confirmed S. aureus breast abscess within one year after delivery identified via needle drainage, incision and drainage, operative intervention, or spontaneous drainage. Women with uncomplicated infectious mastitis as well as a prior history of any S. aureus infection were excluded from the case definition. We selected population-based controls of pregnant women who delivered at our center (N = 32,770). Exclusion criteria for control selection included pre-partum breast abscess, neonatal demise within 24 hours of delivery, stillbirth, and culture-confirmed S. aureus infection at another body site. Controls were matched to cases in a 3:1 fashion by delivery date to minimize potential bias due to secular trends in diagnosis or management of breast abscesses. Controls with a history of any prior S. aureus infection were excluded from the case-control analysis. ## Data collection Hospital databases (Infection Control, Microbiology, Obstetrics, Admission Discharge Transfer, Fiscal databases) were used to extract data for the full population-based cohort, including inpatient admissions, laboratory tests, and radiologic studies. If hospital cost data were not available, we estimated components of medical costs using the Medicare Fee schedule . Additional variables not available electronically were abstracted via medical record review for matched cohort study patients for up to one year after delivery or at the first visit for a subsequent pregnancy, whichever came first. Information was collected on follow-up outpatient visits to obstetrics and gynecology, internal medicine, dermatology, infectious diseases and allergy. Additional information was also collected about relevant radiographic studies (breast ultrasounds, mammograms, and breast magnetic resonance imaging), laboratory testing (complete blood counts (CBC), chemistries, wound and blood cultures), pathology (breast biopsy), central venous catheter placement, and antibiotic use and type, including use of outpatient intravenous antibiotics. Overall hospital readmission rates were collected for the entire birth cohort. Associated adverse outcomes were extracted via chart review for matched cohort study patients only (breastfeeding cessation, milk fistula formation, and adverse effects of antibiotic use (rash, allergy)). # Data analysis Health outcomes, health services utilization, and medical costs were evaluated for both the full cohort and the matched cohort. Descriptive analyses were performed using proportions, means, and medians. We compared rates of outcomes, health service utilization and costs using Wilcoxon rank-sum or Chi-squared tests as appropriate. To maximize accuracy and generalizability, the attributable medical cost of post-partum S. aureus breast abscess was estimated in three ways [fig_ref] Table 1: Cost Estimate Methodologies [/fig_ref]. First, total direct medical costs for cases and non-cases in the overall cohort were estimated from hospital fiscal databases with attributable medical costs calculated as the difference between cases and non-cases and averaged across the entire cohort. A second approach was based on estimating the difference in direct medical costs, based on hospital fiscal databases, only for those services potentially associated with S. aureus breast abscess (i.e., readmission, outpatient visits, laboratory testing, and radiology) for cases and non-cases in the overall cohort. Indirect medical costs, such as facility costs including overhead costs and equipment costs, were not available for analysis. Finally, a third approach to estimating attributable total medical costs in the matched cohort study used national estimates of costs for each unit of health service utilization. The attributable costs were then calculated using the difference in the costs for cases versus controls, averaged across the matched cohort population. More specifically, each unit of service (e.g., hospitalization, procedures, laboratory and radiographic testing, outpatient visits) was multiplied by the cost of the unit of service based on the Medicare Fee Schedule and the Physician Fee Schedule . Laboratory costs were based on maximum Centers for Medicare and Medicaid Services reimbursement rates. Medication costs were estimated using the average wholesale price from the pharmacy Red Bookmultiplied by the number of pills required to complete a standard antibiotic course. Medical costs were adjusted to 2012 dollars using the medical aspect of the gross domestic product deflator, available from the United States Bureau of Labor Statistics. Attributable cost was calculated by determining the mean medical cost of cases and subtracting the mean medical cost of control patients. Pre-specified sub-analysis. We compared health services utilization and medical costs in case patients with MSSA to case patients with MRSA using descriptive statistics, as outlined above. A p-value of ,0.05 was considered statistically significant. Data were analyzed using SAS version 9.3 (SAS Institute, Cary NC). # Ethical considerations Institutional Review Board approval from BIDMC was obtained prior to data collection and analysis. Due to the retrospective study design, and acquisition of data through medical record review, waiver of informed consent was granted. # Results ## Study population A total of 32,770 women delivered at our center during the study period. The mean maternal age among all women delivering at our center was 32.4 years, and 48.1% were primiparous. Baseline demographic description of the entire cohort including the 54 cases and 162 matched controls as well as the epidemic curve were previously published [bib_ref] Risk factors for Staphylococcus aureus postpartum breast abscess, Branch-Elliman [/bib_ref]. The cases included were 30 patients with MRSA abscess and 24 with MSSA breast abscess. The median time to diagnosis of S. aureus breast abscess after postpartum discharge was 34 days (Interquartile range (IQR), 24-49 days). There was no effect of MRSA or cluster period on time to clinical diagnosis. During the cluster period (10/2008-3/2010), a total of 31 patients were diagnosed with culture-confirmed S. aureus breast abscess; 6 with MSSA infection and 25 with MRSA infection. The predominant MRSA strain found during the cluster period was pulsed-field type USA 300-0114 (data not shown) [bib_ref] Medicare and Medicaid programs: hospital outpatient prospective payment; ambulatory surgical center payment;..., Wise [/bib_ref]. Of the full cohort of 32,770 deliveries, 98.2% (32,188/32,770) returned to our center for any type of care during the one-year period following delivery. Among patients enrolled in the matched cohort study, 98.6% (213/216) patients received care at our facility during the one-year period after delivery. ## Outcomes Matched cohort study. The readmission rate among case patients was 50%, compared to less than 2% among control patients [fig_ref] Table 2: Health Services Utilization and Clinical Outcomes Among Women During the Year After... [/fig_ref]. The overall rate of breastfeeding cessation among breast abscess cases was 41%, and did not differ between MRSA and MSSA infections [fig_ref] Table 3: Health Services Utilization and Clinical Outcomes [/fig_ref]. Data regarding breastfeeding cessation was unavailable for control patients. Among all breast abscess cases, six patients developed milk fistulae. The rate of breastfeeding cessation among milk fistula cases was high (66.7%) but not significantly different from all other breast abscess cases. ## Health services utilization Full cohort. The overall rate of readmission among patients in the full cohort who returned to our center (N = 32,188) was 3.2%, and in cases (N = 54), 50% (p,0.0001). Matched cohort study. Cases in the matched cohort study had significantly higher rates of physician visits, radiology utilization and antibiotic utilization than controls without post-partum breast abscess [fig_ref] Table 2: Health Services Utilization and Clinical Outcomes Among Women During the Year After... [/fig_ref]. Across all cases, the median number of ultrasounds per patient was 3.5, range 0-17. Forty-one percent of cases (22/54) had greater than or equal to five breast ultrasounds. For controls, the median number of breast ultrasounds was zero. The rate of mammography was similar in case and control patients. No patients in the matched cohort study received breast magnetic resonance imaging. The majority (75.9%) of the 54 breast abscess cases were treated with needle-guided drainage [bib_ref] Acute puerperal breast abscesses: US-guided drainage, Karstrup [/bib_ref] [bib_ref] Needle aspiration of breast abscesses, Schwarz [/bib_ref] [bib_ref] Management of lactational breast abscesses, Eryilmaz [/bib_ref]. The median number of drainage procedures was two (interquartile range, 1.0-4.0), with a maximum of 15 drainage procedures in one patient. Thirteen (24%) required greater than or equal to five drainage procedures. Six (11%) had surgical incision and drainage; one occurred in the operating room. 14.8% of case patients (8/54) had spontaneous abscess drainage. Antibiotic utilization was available for 92.5% (50/54) case patients. Ninety-eight percent (49/50) of case patients received antimicrobial therapy. In total, 16 different types of antibiotics were prescribed, including two (doxycycline, linezolid) that have Direct medical costs include those directly related to services provided, such as inpatient stay, outpatient office visits, laboratory and radiographic testing. [bib_ref] Wound infection after caesarean section, Moir-Bussy [/bib_ref] Indirect medical costs include facility operating costs, such as building costs, electricity costs, and costs of equipment. [bib_ref] A descriptive study of mastitis in Australian breastfeeding women: incidence and determinants, Amir [/bib_ref] ''All'' indicates that all direct medical costs, including those that may not be related to a diagnosis of post-partum breast abscess were included in the estimate of cost. ''Potentially relevant'' indicates that only direct medical costs related to potentially relevant services (such as visits to internal medicine, obstetrics, infectious diseases, and radiology) were included in the estimate of cost. ''Disease-attributable'' indicates that only services attributable to the diagnosis of post-partum breast abscess were included in the estimate of cost. doi:10.1371/journal.pone.0073155.t001 unsafe or unknown safety profiles in breastfeeding women [bib_ref] Excretion of antimicrobials used to treat methicillin-resistant Staphylococcus aureus infections during lactation:..., Mitrano [/bib_ref] [bib_ref] Antibiotic use in pregnancy and lactation: what is and is not known..., Nahum [/bib_ref]. ## Costs For the entire cohort, the mean attributable cost based on total direct medical cost was $2,414 (95% CI, $1,458-$3,370) and $2,340 (95% CI, $2027-$2610) based on only including potentially relevant services [fig_ref] Table 4: Healthcare Costs of Post-partum Staphylococcus aureus Breast Abscess Compared to Non-infected Controls [/fig_ref]. In the matched cohort study, the attributable cost estimate for the matched cohort study ranged from a minimum in the partial direct facility costs of $2386 (95% CI, $2,027-$2,745) to a maximum estimate based on Medicare cost of $4,012 (95% CI, $3,443-$4,581) [fig_ref] Table 4: Healthcare Costs of Post-partum Staphylococcus aureus Breast Abscess Compared to Non-infected Controls [/fig_ref]. ## Mrsa vs. mssa cases Outcomes. Approximately 11% (6/54) of post-partum breast abscess cases developed milk fistulae; five patients with MRSA (17%) and one patient with MSSA (4%). In total, 5/54 patients (9.3%) of patients had allergic reactions to antibiotic therapy. All allergic reactions occurred in patients with MRSA infection (5/30, 16.7%) versus MSSA infection (0/24, 0%), however, this difference did not reach statistical significance (p = 0.06) [fig_ref] Table 3: Health Services Utilization and Clinical Outcomes [/fig_ref]. Allergic reactions to antibiotic therapy were primarily due to the use of trimethoprim-sulfamethoxazole and vancomycin in patients with MRSA. Health services utilization. Health services utilization was similar among case patients with MRSA and MSSA breast abscesses [fig_ref] Table 3: Health Services Utilization and Clinical Outcomes [/fig_ref] ; however, MRSA cases had significantly more outpatient visits (median 6.0 versus 3.0) and a higher proportion of infectious diseases consultations (57% versus 8.3%, p,0.0001). There were no significant differences in readmission rates or duration of readmissions between MRSA and MSSA cases. Among MRSA cases, there was a trend toward a higher rate of outpatient parenteral antibiotic therapy (10% versus 0%) and number of different antibiotics prescribed (2.6 versus 2.1); however, these differences were not statistically significant. Costs. Despite an increase in both physician visits and antimicrobial usage, there was no significant cost difference between patients with MRSA infection and those with MSSA infection, or between patients who had milk fistulae compared to those who did not [fig_ref] Table 5: Attributable Healthcare Costs of MRSA Cases Compared to MSSA Cases [/fig_ref]. Attributable costs were similar regardless of methodology used. # Discussion Our study is the first to comprehensively evaluate the outcomes and health and economic burdens of post-partum S. aureus breast abscess. In general, we found that the consequences of this infection in a generally healthy population are substantial. According to ambulatory medical records, many women who developed post-partum breast abscess chose to stop breastfeeding due to their infection (41%). In addition, a high proportion of patients with post-partum S. aureus breast abscess required inpatient readmission (50%) and treatment with intravenous antibiotics. Patients with MRSA breast abscess showed a trend towards increased allergic reactions to antibiotic therapy due to the prevalent use in this group of trimethoprim-sulfamethoxazole and vancomycin, drugs that have been associated with among the highest estimated number of emergency department visits per 10,000 outpatient prescriptions [bib_ref] Emergency department visits for antibiotic-associated adverse events, Shehab [/bib_ref]. Notably, in the same study sulfonamide use was associated with a significantly higher rate of moderate-to-severe allergic reactions, compared with all other antibiotic classes combined (4.3% [95% CI, 2.9%-5.8%] vs. 1.9% [95% CI, 1.5%-2.3%]) [bib_ref] Emergency department visits for antibiotic-associated adverse events, Shehab [/bib_ref]. In the BIDMC cohort, the majority of patients received ultrasound-guided drainage for definitive management of their infection rather than open drainage. Multiple previous studies have demonstrated the safety and efficacy of ultrasound-guided needle drainage in the management of breast abscess [bib_ref] Acute puerperal breast abscesses: US-guided drainage, Karstrup [/bib_ref] [bib_ref] Needle aspiration of breast abscesses, Schwarz [/bib_ref] [bib_ref] Ultrasound-guided drainage of breast abscesses: results in 151 patients, Christensen [/bib_ref] [bib_ref] Percutaneous management of breast abscesses. An experience of 39 cases, Berna-Serna [/bib_ref] [bib_ref] Treatment of breast abscesses with ultrasound-guided aspiration and irrigation in the emergency..., Ozseker [/bib_ref] , and that MRSA infections can be successfully treated with needle drainage [bib_ref] Methicillin-resistant Staphylococcus aureus infections may not impede the success of ultrasound-guided drainage..., Chen [/bib_ref]. Few patients (6/45) in our study required surgical incision and drainage for management, and only one required incision and drainage in the operating room, which is similar to rates in previous studies [bib_ref] Management of lactational breast abscesses, Eryilmaz [/bib_ref] [bib_ref] Aspiration of breast abscess under ultrasound guidance: outcome obtained and factors affecting..., Elagili [/bib_ref]. A significant proportion of patients in our study required five or more drainage procedures, and some up to 17 prior to resolution, which is higher than has been found in other studies [bib_ref] Management of lactational breast abscesses, Eryilmaz [/bib_ref] [bib_ref] Aspiration of breast abscess under ultrasound guidance: outcome obtained and factors affecting..., Elagili [/bib_ref] [bib_ref] Treatment of breast abscesses with sonographically guided aspiration, irrigation, and instillation of..., Leborgne [/bib_ref]. There was no association between MRSA infection and requirement for additional drainage procedures, which is consistent with findings in other investigations [bib_ref] Methicillin-resistant Staphylococcus aureus infections may not impede the success of ultrasound-guided drainage..., Chen [/bib_ref]. We found no association between surgical incision and milk fistula formation. In fact, five of the six patients who developed milk fistulae underwent ultrasound-guided needle aspiration of their abscesses only; prior work has demonstrated that milk fistulae occur rarely in this setting [bib_ref] Treatment of breast abscesses with ultrasound-guided aspiration and irrigation in the emergency..., Ozseker [/bib_ref] [bib_ref] Milk fistula: an unusual complication of breast biopsy, Barker [/bib_ref] [bib_ref] Milk fistula: a complication after core breast biopsy, Schackmuth [/bib_ref]. The attributable medical cost of post-partum S. aureus breast abscess was high, and in the same range as other post-partum infections, including surgical site infection after Cesarean delivery ($3761, 95% CI, $3309-$4275) and post-partum endometritis ($4216, 95% CI, $3710-$4792). Medical costs were also similar to surgical site infections following breast surgery ($4967, 95% CI, $3447-$6719), all adjusted to 2012 dollars [bib_ref] Comparison of costs of surgical site infection and endometritis after cesarean delivery..., Olsen [/bib_ref] [bib_ref] Hospital-associated costs due to surgical site infection after breast surgery, Olsen [/bib_ref]. To estimate medical cost, we utilized multiple methods, including an evaluation of total hospital direct cost, partial hospital direct cost, and a Medicare-based cost estimation to improve the accuracy and generalizability of our findings; all methods yielded similar results. The hospital-based estimates of cost may have been lower than the Medicare cost estimate because indirect hospital costs were not available for inclusion in the analysis [fig_ref] Table 1: Cost Estimate Methodologies [/fig_ref]. Interestingly, despite the fact that MRSA cases had significantly more outpatient visits and a higher proportion of infectious diseases consultations, as well as trends toward a higher rate of outpatient parenteral antibiotic therapy and allergic reactions to antibiotic treatment, we found no increase in cost associated with MRSA breast abscess when compared to MSSA infection, perhaps due to limited power to detect a difference between the two groups. Alternatively, outpatient management of these infections may have averted more costly readmissions or procedures [bib_ref] Savings from outpatient antibiotic therapy for osteomyelitis, Eisenberg [/bib_ref]. MRSA patients and MSSA patients did not differ significantly in rates of readmission and readmission length of stay. Our results should be viewed in the context of published data on other types of S. aureus infections that have yielded inconsistent results as to the importance of methicillin-resistance in changing economic costs [bib_ref] Nosocomial methicillin-resistant and methicillin-susceptible Staphylococcus aureus primary bacteremia: at what costs?, Abramson [/bib_ref] [bib_ref] Clinical and financial outcomes due to methicillin resistant Staphylococcus aureus surgical site..., Anderson [/bib_ref] [bib_ref] The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes:..., Cosgrove [/bib_ref] ; [bib_ref] Are there differences in hospital cost between patients with nosocomial methicillin-resistant Staphylococcus..., Ben-David [/bib_ref]. # Limitations All data were collected from a single large birth cohort at a tertiary academic referral center and thus included patients with the most severe complications of post-partum breast abscess, such as milk fistulae. Thus, our experiences and cost analysis may not be generalizable to all practices or settings. To improve the generalizability of our findings, we used multiple methods to estimate attributable medical costs, including an evaluation of total and partial direct hospital costs, as well as an analysis using Medicare estimates. Medicare costs are not directly applicable to a post-partum population; however, Medicare reimbursement has been used in many analyses of healthcare costs . Although practices may have changed during the study period, all cases were matched to contemporary controls, which should control for changes in hospital practice. The patients included in our study were all women of childbearing age, a traditionally young and healthy population. Our prior work demonstrated that the effect of maternal age on risk of post-partum breast abscess in the BIDMC cohort was very small (OR 1.08 per year), therefore, we did not control for age as a potential confounder in medical cost in this generally young and healthy population [bib_ref] Risk factors for Staphylococcus aureus postpartum breast abscess, Branch-Elliman [/bib_ref]. During our study period, there was a large cluster of healthcareassociated, community-onset infections with MRSA USA300-0114. As there was no significant difference in overall health services utilization or medical cost between MRSA and MSSA cases, this likely did not affect our overall findings. However, our results may not be reflective of all MRSA infections, but USA300-0114 MRSA infections in particular. Additionally, although 98.2% of the patients evaluated in our study followed up at our center, we did not have complete follow up data on the entire cohort. It is therefore possible that some health services utilization and costs were not included. Further, in our evaluation of total and partial direct costs, we only included patients with follow up at our center. # Conclusions Post-partum S. aureus breast abscess frequently led to breastfeeding cessation and was associated with additional poor patient outcomes, high health services utilization, and significant attributable medical costs; the economic burden was similar for MRSA and MSSA infections. Prevention efforts should be focused on prevention of all types of S. aureus breast abscess -not just MRSA. Further investigation is warranted to determine the optimal means of preventing S. aureus breast infections during the post-partum period. Includes all direct healthcare costs during the one-year period following post-partum discharge. [bib_ref] Prevalence of and risk factors for methicillin-resistant Staphylococcus aureus colonization and infection..., Maraqa [/bib_ref] Includes only potentially relevant healthcare costs, such as inpatient readmission, relevant outpatient office visits, radiology, and laboratory costs. [bib_ref] Wound infection after caesarean section, Moir-Bussy [/bib_ref] Estimated by multiplying the number of units of each relevant healthcare service by the Medicare reimbursement level for that service. doi:10.1371/journal.pone.0073155.t005 [table] Table 1: Cost Estimate Methodologies. HSU = Health services utilization. Cost per service based on Medicare Fee Schedules. [/table] [table] Table 2: Health Services Utilization and Clinical Outcomes Among Women During the Year After Post-Partum Discharge: Matched Cohort Study Results. [/table] [table] Table 3: Health Services Utilization and Clinical Outcomes: Comparing MRSA to MSSA Cases of Post-partum Staphylococcus aureus Breast Abscess During the Year after Post-Partum Discharge. Wilcoxon rank-sum test, Chi-square, or Fisher's Exact Test used as appropriate. Data missing for 5/54 patients. Includes only severe adverse allergic reactions, including hives and severe skin rash. Minor complications excluded. Includes relevant office visits to obstetrics and gynecology (excluding routine post-partum care, preventive care, and visits for contraception), infectious diseases, breast surgery, internal medicine, and dermatology. No patients in the matched cohort study received allergy and immunology outpatient visits. doi:10.1371/journal.pone.0073155.t003 [/table] [table] Table 4: Healthcare Costs of Post-partum Staphylococcus aureus Breast Abscess Compared to Non-infected Controls. Includes all direct healthcare costs based on hospital fiscal databases during the one-year period following post-partum discharge. Includes only potentially relevant healthcare costs based on hospital fiscal databases, such as inpatient readmission, relevant outpatient office visits, radiology, and laboratory costs. Estimated by multiplying the number of units of each relevant healthcare service by Medicare reimbursement for each service. doi:10.1371/journal.pone.0073155.t004 [/table] [table] Table 5: Attributable Healthcare Costs of MRSA Cases Compared to MSSA Cases. MRSA (N = 30) v. MSSA (N = 24) P-value Total Hospital Direct Costs 1 (Mean, 95% CI) Partial Hospital Direct Costs 2 (Mean, 95% CI) Medicare Estimate of Costs 3 (Mean, 95% CI) [/table]

Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa [formula] CH2690.A CH2690.B CH2690.C ESP058.A ESP058.B ESP058.C ESP067.A ESP067.B ESP067.C F1997.A F1997.B F1997.C MHH17704.A MHH17704.B MHH17704.C Psae1439.A Psae1439.B Psae1439.C ZG8038581181.A ZG8038581181.B ZG8038581181.C CH4681.A CH4681.B CH4681.C F1764.A F1764.B F1764.C F2020.A F2020.B F2020.C MHH16050.A MHH16050.B MHH16050.C MHH16563.A MHH16563.B MHH16563.C MHH17546.A MHH17546.B MHH17546.C Psae1837.A Psae1837.B Psae1837.C 0e+00 5e+06 1e+07 −1e+07 −5e+06 0e+00 [/formula] ## Supplementary preprocessing step parameter value explanation feature detection method centWave peak finding algorithm based on continuous wavelet transformation ppm 15 allowable m/z deviation in consecutive scans, expressed in parts per million minimum peak width 10.9 chromatographic peak widths in s maximum peak width 31.12 signal/noise threshold 10 minimum signal-to-noise ratio mzdiff 0.0155 minimum absolute m/z difference for overlapping chromatographic peaks integration method 1 based on Mexican hat filtered data prefilter peaks 3 minimum number of peaks with at least "prefilter intensity" to be retained after prefiltering prefilter intensity 100 minimum intensity of "prefilter peaks" (see above) noise filter 0 not necessary for centroided data

Predictors of competing mortality to invasive breast cancer incidence in the Canadian National Breast Screening study Background: Evaluating the cost-effectiveness of breast cancer screening requires estimates of the absolute risk of breast cancer, which is modified by various risk factors. Breast cancer incidence, and thus mortality, is altered by the occurrence of competing events. More accurate estimates of competing risks should improve the estimation of absolute risk of breast cancer and benefit from breast cancer screening, leading to more effective preventive, diagnostic, and treatment policies. We have previously described the effect of breast cancer risk factors on breast cancer incidence in the presence of competing risks. In this study, we investigate the association of the same risk factors with mortality as a competing event with breast cancer incidence.Methods:We use data from the Canadian National Breast Screening Study, consisting of two randomized controlled trials, which included data on 39 risk factors for breast cancer. The participants were followed up for the incidence of breast cancer and mortality due to breast cancer and other causes. We stratified all-cause mortality into death from other types of cancer and death from non-cancer causes. We conducted separate analyses for cause-specific mortalities.Results: We found that "age at entry" is a significant factor for all-cause mortality, and cancer-specific and noncancer mortality. "Menstruation length" and "number of live births" are significant factors for all-cause mortality, and cancer-specific mortality. "Ever noted lumps in right/left breasts" is a factor associated with all-cause mortality, and non-cancer mortality.Conclusions: For proper estimation of absolute risk of the main event of interest common risk factors associated with competing events should be identified and considered. # Background Women in a clinical trial with breast cancer incidence as the endpoint may die due to causes other than breast cancer before the occurrence of the cancer. Competing events should be taken into account in evaluating the efficacy and cost-effectiveness of screening interventions both at the population level and for a given individual (personalized screening regimes). A specific screening intervention may be recommended for a woman based on her age and other characteristics, such as having a family history of breast cancer. However, these characteristics can also affect the occurrence of competing events; so, it is essential to study the effect of risk factors on both breast cancer incidence and its competing events such as mortality due to other causes. Some studies have focused on the main event of interest and considered competing risks. Fang et al. [bib_ref] Hospitalization for osteoarthritis and prostate cancer specific mortality among Swedish men with..., Fang [/bib_ref] examined the potential role of nonsteroidal anti-inflammatory drugs on prostate cancer specific mortality while controlling for other competing causes of death. Yi et al. [bib_ref] Meric-Bernstam F: Factors affecting the decision of breast cancer patients to undergo..., Yi [/bib_ref] determined the factors associated with a contralateral prophylactic mastectomy while taking into account the competing risk of the recurrence of the primary breast cancer. Mell et al. [bib_ref] Predictors of Competing Mortality in Advanced Head and Neck Cancer, Mell [/bib_ref] identified predictors of non-cancer causes of death in head and neck cancer and developed a risk stratification model for these competing events. Mell et al.used competing risk modeling to identify predictors of non-cancer mortality in women with early breast cancer, while considering disease recurrences as competing risks. Other studies focused on all competing events and investigated the effect of some risk factors on the main event of interest and its competing risks. Several studies used cohort life tables to derive probability estimates for death due to breast cancer as well as mortality due to other causes. Hence, they were able to estimate reductions in breast cancer mortality [bib_ref] Competing risks to breast cancer mortality, Rosenberg [/bib_ref] [bib_ref] Competing risks to breast cancer mortality in Catalonia, Vilaprinyo [/bib_ref]. Lambert et al. [bib_ref] Quantifying differences in breast cancer survival between England and Norway, Lambert [/bib_ref] estimated and partitioned the crude probability of allcause mortality to the probabilities due to cancer and other causes. Crude probabilities can be used to understand the impact of disease on individual patients and help assess different treatment options. Daskivich et al. [bib_ref] Comorbidity and competing risks for mortality in men with prostate cancer, Daskivich [/bib_ref] assessed the competing risks of mortality from nonprostate cancer on patients with prostate cancer. Barnes et al. [bib_ref] Population attributable risk of invasive postmenopausal breast cancer and breast cancer subtypes..., Barnes [/bib_ref] emphasized that some risk factors associated with breast cancer have been shown to affect the risk of other health outcomes, so competing risks and benefits may influence public health policy decisions. Vilaprinyo et al. [bib_ref] Competing risks to breast cancer mortality in Catalonia, Vilaprinyo [/bib_ref] estimated the risk of death from causes other than breast cancer; used for assessing the effect of mammography screening on breast cancer mortality. In another study [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] , we have investigated the association of 39 risk factors with breast cancer incidence in the presence of competing risk. We used data from the Canadian National Breast Screening Study (CNBSS) in which the information on risk factors were collected at enrolment or recorded by a nurse or physician at the initial physical examination of the breasts. In the CNBSS, women were enrolled alive and had to be cancer-free. They were randomly assigned to study and control groups and were followed up for breast cancer incidence and mortality. By the end of the study period, a woman might have been diagnosed with breast cancer, died from any causes including breast cancer, or remained alive and cancer-free. A schematic illustration of the three possible terminating points is shown in [fig_ref] 1: Invasive breast cancer [/fig_ref]. The main focus of the current study and of [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] is on breast cancer incidence and its risk prediction model. Therefore, in these two studies, we terminate the followup of a woman as soon as she is diagnosed with breast cancer. Thus, we do not study the progression of a tumor from diagnosis to death. Breast cancer incidence and mortality due to nonbreast cancer causes are competing risks and both should be analyzed. The necessity of studying both events is given in detail in Discussion section of the paper. In this paper, we investigate the effect of 39 risk factors on mortality due to causes other than breast cancer. So, the current paper and [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] are complimentary. We identify the factors common to both breast cancer incidence and competing mortality. In addition, we stratify all-cause mortality (excluding breast cancer), comparing it to death caused by other types of cancer and death from non-cancer causes. We conduct separate analyses for all-cause mortality and the two cause-specific mortalities. The reason for stratification is to examine whether any of the risk factors are particularly associated with both breast cancer incidence and cancer mortality. The findings in this case are more informative and help us understand better the biological mechanism through which a risk factor influences cancer and non-cancer mortality. # Methods ## Study population and period The Canadian National Breast Screening Study (CNBSS) has been described previously [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] [bib_ref] Canadian national breast screening study: 1. Breast cancer detection and death rates..., Miller [/bib_ref] [bib_ref] Canadian national breast screening study: 2. Breast cancer detection and death rates..., Miller [/bib_ref]. Its main objective is to assess the effect of mammography in reducing breast cancer mortality. The study consists of two randomized controlled trials of 89,835 women; 50,430 aged 40-49 and 39,405 aged 50-59. The women were recruited at 15 Canadian centres between 1980 and 1985. All participants signed an informed consent form developed with approval from the University of Toronto Human Experimentation Committee when they enrolled in the CNBSS which included explicit agreement for linkage to vital statistics records and analysis of the data in the future. Women in the age group 40-49 were randomly selected to receive either an annual mammogram and physical examination (intervention group) or only an initial physical examination with no mammography (control group). Women aged 50-59 were randomly selected to receive either an annual mammogram and physical examination (intervention group) or an annual physical examination only (control group). A flow diagram of the CNBSS is presented in [fig_ref] Figure 2: A flow diagram of the CNBSS [/fig_ref]. Woman in the CNBSS were not pregnant at the time of enrolment, had no history of breast cancer and no mammogram in the past 12 months. At enrolment, they completed enrolment and epidemiology questionnaires which included information on demographics, life style, family history of breast cancer, and personal history of breast disease. Moreover, at enrolment a nurse or physician performed a physical examination of the breasts and recorded information on several risk factors. The CNBSS contains information on breast cancer diagnoses reported for women in the intervention group; these women received up to five mammograms. In addition, breast cancer diagnoses were identified by record linkage with the National Cancer Registry and deaths through linkage with the Canadian Mortality Database at Statistics Canada for both the control and the intervention groups after the annual screenings had ended. For this analysis we consider 1980-1989 as the study period and exclude breast cancers which were diagnosed less than six months after enrolment to eliminate longterm prevalent breast cancers. A total of 89,434 women were considered in our study: of these, 944 were diagnosed with invasive breast cancer, 922 died from causes other than breast cancer [fig_ref] Table 1: Causes of deaths [/fig_ref] describes the causes of death for these 922 women), and 87,568 were neither diagnosed with breast cancer nor had died from other causes by the end of 1989. Of the 922 who died from causes other than breast cancer, 536 died from cancers other than breast cancer, and 386 deaths were due to non-cancer causes. ## Risk factors and data preparation This study considers 39 risk factors (see [bib_ref] Competing risks to breast cancer mortality, Rosenberg [/bib_ref] collected at enrolment or recorded by a nurse or physician at the initial physical examination of the breasts. We classify these factors into four groups: sociodemographic factors, reproductive factors, lifestyle and health behaviours, and history of breast disease. For a premenopausal woman, "menstruation length" is the difference between her "age at entry" and "age at menarche", and for a postmenopausal woman, it is the difference between her "age at the last menstrual period" and "age at menarche". Less than 5 % of the values of all data were missing and have been imputed. A normal linear regression model is used to impute missing values in the continuous variables. Missing values in the categorical variables are imputed using a logistic or generalized logistic model. Each woman is given a score for families/ relatives with breast cancer. Each relative, depending on her degree (first, second, third, fourth, fifth degree and above) makes a contribution of 2 6Àdegree number ð Þ to the score value. For example, a woman with one first degree and one second degree relative with breast cancer is given a score of 2 6À1 ð Þ þ 2 6À2 ð Þ ¼ 48. A woman with no relatives with breast cancer receives a score of 1 [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref]. The calculation method for score value is adapted from the U.S. Preventive Services Task Force recommendations on genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility. # Statistical method In this study, we use multivariate cause-specific Cox regression analysis to investigate the associations of the 39 risk factors with all-cause mortality, cancer-specific mortality, and non-cancer mortality. Incidence of breast cancer is considered a competing event in the analysis of all-cause mortality. In our analysis of the cause-specific mortality from cancer and non-cancer causes, mutually exclusive events are the competing events. For example, breast cancer incidence and non-cancer mortality are considered events competing with cancer-specific mortality. It should be noted that hazard of subdistribution proposed by Fine and Gray [bib_ref] A proportional hazards model for the subdistribution of a competing risk, Fine [/bib_ref] is another method for regression modeling of an event in the presence of competing risks. The hazard of subdistribution can be used for directly modeling the effect of covariates on the cumulative risk of the event of interest. However, a cause-specific model must be used when the goal is to investigate the biological effect of risk factors on the event of interest. We also fitted the Fine-Gray model to our data and obtained very similar results to those obtained from the cause-specific hazard model. Results from the hazard of subdistribution model are not shown in this paper due to space limitations. We use the procedure PHREG in SAS v. 9.3 (SAS Institute, Cary, NC) to build regression models. To construct the models we first conduct univariate analysis of each individual variable, we then combine the significant factors in a multivariate model to adjust the risk for all significant factors. Moreover, we perform a backward model selection to recheck the final model. We considered a variable (risk factor) statistically significant if its probability (P) value was less than 0.05. We checked the interaction of the variables with each other and with time to event (time of death or censoring time) to find those variables with a time-varying effect on the risk of mortality. No interaction terms were found to be statistically significant. In , we used a non-parametric method formulated by Kalbfleisch and Prenticeto estimate cause-specific hazard and obtained a discrete estimate of the cumulative incidence function (cumulative risk). For this purpose, we used function cuminc in package cmprsk in R (http://cran.r-project.org/web/packages/cmprsk/cmprsk. pdf). # Results From 89,434 participants in the study, 1.06% (N = 944) were diagnosed with invasive breast cancer, 1.03% (N = 922) died from causes other than breast cancer, and 97.9 % (N = 87,568) were alive and not diagnosed with breast cancer by the end of 1989. 58.1% (N = 536) of the deaths were due to other types of cancer, and 41.9% (N = 386) due to causes other than cancer. The mean times from enrolment to cancer diagnosis and to death are 3.52 (SD 2.0) and 4.26 (SD 2.17) years respectively. The median from enrolment to cancer diagnosis and to death are respectively 3.18 and 4.23 years. The mean and median follow-up time of women in the censored group (alive and cancer-free by the end of the study) are 6.72 (SD 1.32) and 6.39 years, respectively. .A shows the observed ten-year cumulative risk of invasive breast cancer and all-cause mortality (excluding breast cancer death) stratified by two age-groups 40-49 and 50-59. The risk of breast cancer incidence is always higher than the risk of all-cause mortality for younger women (40-49) within the follow-up period. However, for older women (50-59), although breast cancer incidence is lower than the risk of death in about the first six years of follow-up; the curves for breast cancer incidence and all-cause mortality cross at this point and the probability of death is higher than breast cancer incidence subsequently. .B shows the ten-year cumulative risk of breast cancer incidence, cancer-specific mortality, and non-cancer mortality stratified by the two age- groups. When we compare the breast cancer and mortality curves in both age groups, we see that the women in our study always have a higher probability of breast cancer incidence than cancer-specific and non-cancer mortality. Moreover, although for women aged 40-49 the risk of death from cancer and noncancer causes is similar in the first two and half years of follow-up, death from cancer is more likely to occur than death from non-cancer causes afterwards. For women in the age-group 50-59, death from cancer is more likely to occur than non-cancer mortality in the entire follow-up period. The probability estimates and their standard errors for breast cancer incidence, cancer mortality, and non-cancer mortality at eight years after follow-up are 0.013 (0.00047), 0.007 (0.00037), and 0.006 (0.00033) respectively, for the overall population. Predictors of All-cause Mortality [fig_ref] Table 6: Adjusted risk for all statistically significant factors for all-cause mortality excluding breast... [/fig_ref] presents the factors found to be statistically significant for all-cause mortality. The reference levels of the categorical variables are also shown in the table. As an example, "1-2 children" is the reference level for "number of live births". For continuous variables, such as age or menstruation length, the hazard ratio is the change in the hazard for a one-unit increase in the value of the variable, holding all other variables constant. The results show that older women have higher risk of all-cause mortality (HR 1.12, 95% CI 1.10-1.13). For example, compared to a 40-year old woman with the same characteristics, a woman aged 50 at enrolment is almost three times (1:118 10 % 3:00) more likely to die due to any causes at any given point in time. Taking estrogen or progesterone supplements for a longer time and having more years of menstruation are slightly protective factors against all-cause mortality. There is no evidence that a nulliparous woman is more likely to die due to any causes than a woman with one or two live births; however, having more than two live births decreases the probability of allcause mortality (HR 0.84 and 0.76 for three-four and more than four live births, respectively). Breast self examination (BSE) is another protective factor for all cause mortality (HR 0.83, 95% CI 0.73-0.94). Although smoking less than 11 cigarettes per day does not statistically significantly increase all-cause mortality, smoking 11 or more cigarettes per day significantly increases the probability of death due to any causes. For example, a woman who smokes more than 50 cigarettes per day is four times more at risk of death than a non-smoker. Women who have ever noted lumps in their breasts or have had discharge from their breasts have respectively 1.3 and 1.5 times higher risk of death than those who have not. Having the first child at age 14 to 19 is associated with a 1.4-fold higher risk of death (HR 1.41, 95% CI 1.14-1.74) than having the first child at age 20 to 25. ## Predictors of cancer-specific mortality The predictors for mortality due to cancer (excluding breast cancer) are presented in [fig_ref] Table 7: Adjusted risk for all statistically significant factors for cancer-specific mortality [/fig_ref]. "Age at entry", "length of estrogen/progesterone used", "menstruation length", "breast self examination", and "having the first child birth at age 14-19" are factors having a similar or the same effect on the risk of all-cause mortality and mortality due to cancer. Women who have a bi-lateral oophorectomy are 1.3 times more at risk of death due to cancer than those who do not. As for all-cause mortality, there is no evidence that a nulliparous woman is more likely to die due to cancer than a woman with one or two live births, but having more than two live births decreases the probability of cancer death (HR 0.74 and [fig_ref] Table 8: Adjusted risk for all statistically significant factors for non-cancer mortality Ref * [/fig_ref] presents the factors found to be statistically significant for non-cancer mortality. The table shows that "age at entry" and "length of estrogen/progesterone used" have similar effects on both non-cancer mortality and all-cause mortality. In addition, women whose ethnic origin was reported as not being Canadian have a 5-fold (HR 0.22, 95% CI 0.10-0.46) lower risk of noncancer mortality than women whose ethnic origin was reported as Canadian. Women who have had a hysterectomy are 1.3 times more likely to die from non-cancer causes than those who have not. Women who reported smoking more than 10 cigarettes per day were found to have a higher risk of non-cancer mortality (HR 1.82, 2.62, and 5.86, respectively, for smoking 11-25, 26-50, and > 50 cigarettes per day). Finally, women who have ever noted lumps in their breasts or have had discharge from their breasts have respectively 1.6 and 2.0 times more risk of non-cancer death than those who have not. compares the significant factors in all three mortality models and the model for breast cancer incidence [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] (factors for breast cancer incidence are reported in detail in [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref]. "Age at entry", "length of estrogen/progesterone used (months)", and "cigarettes smoked per day" are common to the three morality models. If we compare the significant risk factors for mortality with those associated with breast cancer incidence, we find "age at entry" appears in all four models. "Menstruation length" and "number of live births" are statistically significant risk factors for breast cancer incidence, all-cause mortality, and cancer-specific mortality. "Ever noted lumps in breasts" is associated with breast cancer incidence, all-cause mortality, and non-cancer mortality. ## Predictors of non-cancer mortality # Discussion In this study, we identify the predictors for all-cause mortality, cancer-specific mortality (excluding mortality from breast cancer), and non-cancer mortality. As previously reported, mortality due to causes other than breast cancer is a competing risk for breast cancer incidence in the data from the Canadian National Breast Screening Study (CNBSS). It should be noted that we investigated the associations for allocation group (no-mammography vs. mammography) to both breast cancer incidence and its competing mortality. We conducted univariate and multivariate analyses, and in none of the models, did this categorical variable reach statistical significance when combined with other risk factors. This does not mean there is no some real difference in breast cancer incidence in these two groups, but the data do not show it statistically. Otherwise, the analysis would have been stratified by allocation group and separate results obtained for each group. Therefore, to make use of a larger sample size for statistical analysis we combined all the data without stratification by allocation group. We found that using estrogen or progesterone supplements for a longer time and longer menstruation are both protective factors against all-cause mortality. These results are consistent with other studies' findings that mortality among women who use hormones is lower than among nonusers [bib_ref] Postmenopausal hormone therapy and mortality, Grodstein [/bib_ref] [bib_ref] Estrogen Use and All-Cause Mortality Preliminary Results From the Lipid Research Clinics..., Bush [/bib_ref] , and the later the onset of menopause, the longer a woman is likely to live. A negative association of these two factors with mortality could primarily be explained by reducing the occurrence of cardiovascular disease [bib_ref] Estrogen Use and All-Cause Mortality Preliminary Results From the Lipid Research Clinics..., Bush [/bib_ref]. Ten-year cumulative risks by age groups. A, invasive breast cancer and the all-cause mortality. B, invasive breast cancer, the all-cause mortality, cancer-specific and non-cancer mortalities. The all-cause mortality analysis revealed a decrease in mortality with number of live births. In the literature, there is no consistent pattern of mortality with the number of births [bib_ref] The effect of number of births on women's mortality: Systematic review of..., Hurt [/bib_ref]. Some studies show a clear decrease in mortality for women with 2-3 children compared to nulliparous women, and a non-statistically significant increase in the risk for women with more than three children. The result of some other studies is consistent with our result and shows an overall negative association between higher parity and mortality [bib_ref] Reproductive History and Mortality in Late Middle Age among Norwegian Men and..., Grundy [/bib_ref]. Similar to our result which shows an increase in the risk of mortality when a woman has her first child at a younger age [bib_ref] A proportional hazards model for the subdistribution of a competing risk, Fine [/bib_ref] [bib_ref] Postmenopausal hormone therapy and mortality, Grodstein [/bib_ref] [bib_ref] Estrogen Use and All-Cause Mortality Preliminary Results From the Lipid Research Clinics..., Bush [/bib_ref] , Grundy and Kravdal [bib_ref] Reproductive History and Mortality in Late Middle Age among Norwegian Men and..., Grundy [/bib_ref] also found higher mortality for parents who had their first child at a younger age which tended to decrease with older age at first birth. Similar to the results from all-cause mortality, increase in the number of live-births exhibits a decreasing trend in the risk of cancer-specific mortality, although the nulliparous level does not reach statistical significance. As the number of pregnancies increases the risk of cancerspecific mortality increases as well, although only more than ten times of pregnancy reaches statistical significance. Moreover, increasing age at first child birth shows a clear decreasing trend for risk of cancer-specific mortality; however, only decrease in the risk of women who had their first child-birth at age 20-25 compared to those who had it at age 14-19 is statistically significant. We also found that women who were practicing BSE on enrolment also had lower all-cause mortality. Arguably, women who practice BSE are more likely to have better personal health practices such as exercise, nutrition, etc. Event though we observe no trend in the first three levels of the number of cigarettes smoked per day (i.e. up to 11-25 cigarettes per day), the next three levels clearly show an increasing trend in the risk in all three mortality models. Ever noting lumps in the breasts or having discharge from the breasts are both associated with non-cancer mortality. Although we do not have sufficient data to investigate biological association of these two factors with each cause of mortality, one reason for increased risk of non-cancer mortality could be anxiety caused by discovery of a breast abnormality [bib_ref] The Emotional Journey of Women Experiencing a Breast Abnormality, Blow [/bib_ref]. Women who experience the discovery will be coping with fears about the future and dealing with emotional ups and downs [bib_ref] The Emotional Journey of Women Experiencing a Breast Abnormality, Blow [/bib_ref]. By using the CNBSS, our study takes advantage of a large cohort linked to the Canadian Cancer Registry and the Mortality Database at Statistics Canada and relatively complete data collected at the time of enrolment and the initial physical examination of the breasts. This makes our investigation of the association of 39 different risk factors with breast cancer incidence [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] and its competing mortality reliable. Estimating the absolute risk of an event as accurately as possible is fundamental in clinical decision-making [bib_ref] Prognostic Models with Competing Risks Methods and Application to Coronary Risk Prediction, Wolbers [/bib_ref] [bib_ref] On criteria for evaluating models of absolute risk, Gail [/bib_ref] [bib_ref] Actual and actuarial probabilities of competing risks: apples and lemons, Grunkemeier [/bib_ref] [bib_ref] Treatment with drugs to lower blood pressure and blood cholesterol based on..., Jackson [/bib_ref]. In the analysis of data in a competing risks setting, both results for the event of interest and its competing risks should be presented [bib_ref] Cumulative incidence in competing risks data and competing risks regression analysis, Kim [/bib_ref] [bib_ref] Methods of competing risks analysis of end-stage renal disease and mortality among..., Lim [/bib_ref] , and risk factors for each competing risk identified [bib_ref] Cumulative incidence in competing risks data and competing risks regression analysis, Kim [/bib_ref]. The necessity for analysis of both events of interest and their competing mortality can be explained through the definition of absolute cause-specific risk (given in Appendix). Moreover, describing the natural history of cancer and possible pathways that an individual may go through is an essential component of any simulation model which is developed to assess the effectiveness of different intervention policies. In a microsimulation model, the life histories of many individuals are generated, and each history substantially depends on the value of different risk factors for a given individual. For example, a woman who has a relative with breast cancer is at a higher risk of being diagnosed with breast cancer at a younger age. Other competing events, such as death due to different causes can also be influenced by the risk factors. For example, a woman aged 70 or above may die due to heart attack before she is diagnosed with breast cancer. Therefore, the effect of risk factors on all competing events should be studied and described to model realistic life paths of individuals. The simulation models could be either developed for evaluating costs and benefits of cancer interventions at the population level, or for an individual according to her risk factors, as the practice of personalized medicine [bib_ref] Economic evaluation of targeted cancer interventions: Critical review and recommendations, Elkin [/bib_ref]. A cost-effective breast cancer screening policy relies on knowing the absolute risk of breast cancer, and as discussed, to obtain the absolute risk, competing risks should also be studied and taken into account. The absolute risk can then be used to decide whether a woman is categorized as high risk and whether a specific intervention with higher frequency should be recommended. Gail et al. [bib_ref] Projecting individualized probabilities of developing breast cancer for white females who are..., Gail [/bib_ref] developed an online risk assessment tool to estimate a woman's absolute risk of developing invasive breast cancer . In this tool, the absolute risk is estimated based on a woman's risk factors including age, family history of breast cancer, number of biopsies, etc. A woman is identified at high risk if her lifetime risk of invasive breast cancer exceeds 20% [bib_ref] Women at high risk for breast cancer -what the primary care provider..., Afonso [/bib_ref]. Gail's model [bib_ref] Projecting individualized probabilities of developing breast cancer for white females who are..., Gail [/bib_ref] also investigates competing risks, namely the age-specific risk of mortality from causes other than breast cancer by using national mortality rates. # Conclusions In this study, we show that some risk factors are statistically significant for both the main event of interest (incidence of invasive breast cancer) and a competing event (mortality due to non-breast cancer causes). In addition, we identify other risk factors associated with all-cause mortality and cause-specific mortality. All these factors must be taken into account in estimating the probability of a competing event. With more accurate estimation of the probability of a competing event, the estimations of absolute risk of the main event of interest can be estimated more precisely. This in turn leads to more robust cost-effective analysis of preventive, diagnosis, and treatment policies which are decided based on the results of the absolute risk prediction model. We used a cause-specific model to conduct regression analysis on our competing risks data. In addition, we fitted the Fine-Gray model and the results were very close to the results of the causespecific hazard model. The cause-specific model is more clinically and biologically understandable since it directly describes the effect of a covariate on a specific cause and its hazard, regardless the effect of other covariates [bib_ref] Methods of competing risks analysis of end-stage renal disease and mortality among..., Lim [/bib_ref]. In our data, the risk of both breast cancer incidence [bib_ref] Incidence of invasive breast cancer in the presence of competing mortality: The..., Taghipour [/bib_ref] and its competing mortality are minor, for which the Cox cause-specific hazard is more reliable to be used [bib_ref] Methods of competing risks analysis of end-stage renal disease and mortality among..., Lim [/bib_ref]. Moreover, the generality of hazard of subdistribution is restricted to populations with similar characteristics and competing risk rate [bib_ref] Analysing and interpreting competing risk data, Pintilie [/bib_ref] , the latter being relaxed in the cause-specific hazard model. On the other hand, the subdistribution hazard can be used to calculate the cumulative incidence function of the event of interest and to directly interpret the effect of covariates on this function [bib_ref] Tutorial in biostatistics: competing risks and multi-state models, Putter [/bib_ref]. ## Appendix-calculating absolute cause-specific risk When there are K competing causes, the cause-specific hazard, which is defined as the instantaneous probability of failing from cause k is λ k t ð jZÞ ¼ lim Δt!0 P t≤T ≤t þ Δt; C ¼ k ð jT ≥t; ZÞ Δt; ð1Þ where Z is a vector of covariates (risk factors), and the overall hazard is λ t ð jZÞ ¼ P K k¼1 λ k t ð jZÞ. The absolute cause-specific risk or cumulative incidence function (CIF) for cause k is defined as follows [bib_ref] Evaluating health outcomes in the presence of competing risks: a review of..., Varadhan [/bib_ref] : [formula] I k t ð jZÞ ¼ P T≤t; C ¼ k ð Þ¼ Z t o λ k s ð jZÞS s ð jZÞds:ð2Þ [/formula] In equation (2), S t ð jZÞ ¼ P T≥t ð jZÞ ¼ e ÀΛ t ð jZ Þ is the overall survival function and Λ t ð jZÞ ¼ R t 0 λ s ð jZÞds is the cumulative hazard. Therefore, the cumulative hazard is a function of all cause-specific hazards k ¼ 1; . . . ; K . Equation [bib_ref] Meric-Bernstam F: Factors affecting the decision of breast cancer patients to undergo..., Yi [/bib_ref] clearly shows that to obtain the cumulative risk of an event of interest (in our case, breast cancer Comparing the predictors of breast cancer incidence, all-cause mortality, cancer-specific mortality and noncancer mortality Variable Breast cancer incidence All-cause mortality Cancer mortality Non-cancer mortality Age at entry + + + + ## Interaction [fig] 0: Alive and cancer-free [/fig] [fig] 1: Invasive breast cancer(2) All-cause mortality except breast cancer [/fig] [fig] Figure 2: A flow diagram of the CNBSS. [/fig] [table] Table 2: Characteristics of the study cohort: Socio-demographic factors [/table] [table] Table 3: Characteristics of the study cohort: Reproductive factors [/table] [table] Table 4: Characteristics of the study cohort: Lifestyle and health behaviours [/table] [table] Table 5: Characteristics of the study cohort: history of breast disease [/table] [table] Table 6: Adjusted risk for all statistically significant factors for all-cause mortality excluding breast cancer death [/table] [table] Table 7: Adjusted risk for all statistically significant factors for cancer-specific mortality [/table] [table] Table 8: Adjusted risk for all statistically significant factors for non-cancer mortality Ref * : Reference level. [/table]

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic. # Introduction There has been a substantial interest in using microbial fatty acid biosynthesis as a platform for a variety of biorenewable chemicals [bib_ref] Manufacturing molecules through metabolic engineering, Keasling [/bib_ref] [bib_ref] Derivation and synthesis of renewable surfactants, Foley [/bib_ref] [bib_ref] Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced..., Handke [/bib_ref] [bib_ref] From fields to fuels: recent advances in the microbial production of biofuels, Kung [/bib_ref]. However, there are challenges associated with harnessing the fatty acid biosynthesis pathway for producing chemicals at industrially relevant titers, productivities, and yields. For example, it has been noted by multiple researchers that product toxicity is a major problem for the microbial production of carboxylic acids [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref] [bib_ref] Membrane stresses induced by overproduction of free fatty acids in Escherichia coli, Lennen [/bib_ref] [bib_ref] Membrane stress caused by octanoic acid in Saccharomyces cerevisiae, Liu [/bib_ref] [bib_ref] Engineering Escherichia coli to synthesize free fatty acids, Lennen [/bib_ref] [bib_ref] Understanding biocatalyst inhibition by carboxylic acids, Jarboe [/bib_ref]. Microbial tolerance of inhibitors, either present in the plantderived feedstock or a toxic desired product, is a common problem in the fermentative production of biorenewable fuels and chemicals [bib_ref] Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals, Jarboe [/bib_ref] [bib_ref] Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli, Mills [/bib_ref] [bib_ref] Engineering microbial biofuel tolerance and export using efflux pumps, Dunlop [/bib_ref]. Knowing the mechanism of inhibition can enable rational design strategies for addressing tolerance. Transcriptome analysis is one method for identifying these mechanisms [bib_ref] Membrane stress caused by octanoic acid in Saccharomyces cerevisiae, Liu [/bib_ref] [bib_ref] Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals, Jarboe [/bib_ref] [bib_ref] Furfural inhibits growth by limiting sulfur assimilation in ethanologenic Escherichia coli strain..., Miller [/bib_ref] [bib_ref] An integrated network approach identifies the isobutanol response network of Escherichia coli, Brynildsen [/bib_ref]. It is relatively well-established that one of the major effects of short chain carboxylic acid toxicity is membrane damage, largely due to the hydrophobic nature of the carbon chain [bib_ref] Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential, Desbois [/bib_ref]. It is also well-accepted that exogenous challenge with carboxylic acids can cause intracellular acidification, interfering with cellular function and imposing an ATP burden [bib_ref] External concentration of organic acid anions and pH: key independent variables for..., Carpenter [/bib_ref] [bib_ref] The ability of Escherichia coli O157:H7 to decrease its intracellular pH and..., Diez-Gonzalez [/bib_ref] [bib_ref] Perspectives on the use of organic acids and short chain fatty acids..., Ricke [/bib_ref] [bib_ref] Transport of lactate and acetate through the energized cytoplasmic membrane of Escherichia..., Axe [/bib_ref]. Our previous work quantified the effect of octanoic acid (C8) on membrane integrity, fluidity, hydrophobicity and composition [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref] and we concluded that diffusion of octanoic acid into the membrane impairs its function. Here we used transcriptomic analysis of exogenous octanoic acid challenge to identify and quantify other mechanisms of inhibition, as well as exploring strategies for improving tolerance. # Materials and methods ## Strains and growth conditions Escherichia coli strain K-12 MG1655 was obtained from ATCC (Manassas, VA, USA) [fig_ref] Table 1: Strains and plasmids used in this study [/fig_ref]. All strains were grown in 25 mL MOPS minimal media [bib_ref] Gene expression in bacteria using TnphoA and TnphoA' elements to make and..., Wanner [/bib_ref] with 2% dextrose in 250 mL baffled flasks at 37uC. Overnight cultures were diluted to an optical density of 0.05 at 550 nm (OD 550 ) for specific growth measurements and RNA extraction, and diluted to 0.1 for intracellular pH, c-amino butyric acid, and membrane lipid composition measurements. 4 M C8 stock solutions were prepared in 100% ethanol. The concentration of ethanol used in these experiments did not have a significant impact on growth (data not shown). Media with octanoic acid were pH-adjusted to 7.0 with 2 M potassium hydroxide. The specific growth rate was determined by measuring the OD 550 during the exponential phase, as previously described [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. The carboxylic acid production strain ML103 [bib_ref] Effect of acetate formation pathway and long chain fatty acid CoA-ligase on..., Li [/bib_ref] was obtained from Ka-Yiu San (Rice University). This strain was grown in 25 mL M9 with 1.5% dextrose in 250 mL baffled flasks at 30uC with the appropriate antibiotics and inducers, as described below. Plasmids pJDT1 [bib_ref] Export of active green fluorescent protein to the periplasm by the twin-arginine..., Thomas [/bib_ref] ,obtained from Anja Nenninger (University of London), was induced by 200 mM L-arabinose and 100 mg/mL ampicillin was used for maintenance. pCA-cfa and pCA-lrp [bib_ref] Complete set of ORF clones of Escherichia coli ASKA library (A complete..., Kitagawa [/bib_ref] , obtained from Ramon Gonzalez (Rice University), were induced by 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) and 25 mg/mL chloramphenicol was used for maintenence. pZS-GFP was cloned using the Gibson Assembly Cloning kit (New England Biolabs, Ipswich, MA, USA), with pZS [bib_ref] Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and..., Yazdani [/bib_ref] as the backbone and the GFP gene from pJDT1. pZS-GFP was induced by using 50 ng/mL anhydrotetracycline (Fisher Scientific, Hampton, NH, USA) and 25 mg/mL chloramphenicol was used for maintenance. pXZ18Z, obtained from Ka-Yiu San (Rice University), was induced by 100 mM IPTG and 100 mg/mL ampicillin was used for maintenance [fig_ref] Table 1: Strains and plasmids used in this study [/fig_ref]. Transcriptomics sample preparation E. coli MG1655 was grown to midlog (OD 550 ,0.8) with or without 10 mM octanoic acid and cells were harvested for RNA purification. Briefly, cells were harvested by swirling in a dry ice/ ethanol water bath until cold and then centrifuged at 5,000 g, 20 min. at 4uC; the resulting cell pellets were stored in RNA Later solution (Life Technologies, Carlsbad, CA) at 280uC. RNase AWAY solution (Life Technologies) was used to remove contaminating RNase. The RNeasy Mini Kit (Qiagen, Venlo, Netherlands) was used to isolate total RNA, which was then incubated at 37uC with RNaseOut (New England Biolabs) and DNaseI (New England Biolabs) according to the manufacture's protocol for 1 hour. The samples were mixed with saturated phenol/chloroform (pH = 4.5) (Life Technologies) and precipitated with ethanol and 30 mL 3 M sodium acetate (pH = 5.5) (Fisher Scientific) overnight at 280uC. After precipitation, the tubes were centrifuged for 30 min. at [bib_ref] External concentration of organic acid anions and pH: key independent variables for..., Carpenter [/bib_ref] ## Transcriptomic data normalization and analysis Background adjustment, normalization, and summarization calculations were performed in MATLAB v. R2012b (Math-Works, Natick, MA, USA) using GCRMA [bib_ref] Stochastic models inspired by hybridization theory for short oligonucleotide arrays, Wu [/bib_ref]. Using pooled data, permutation t-tests (10,000 permutations) were performed to identify transcripts with statistically significant changes in abundance (q-value ,0.05). Additionally, a fold change (log 2 (treatment/control)) cutoff of 61 was applied to identify genes that demonstrated a sufficient magnitude of variation between experimental conditions. Probes not annotated as MG1655, but orthologous to E. coli strain MG1655 were included. Probes relating to other species were excluded from analysis. Additionally, overrepresentation of Gene Ontology terms associated with significant expression perturbations was examined and visualized using the Cytoscape tool [bib_ref] Cytoscape 2.8: new features for data integration and network visualization, Smoot [/bib_ref] and BiNGO [bib_ref] BiNGO: a Cytoscape plugin to assess overrepresentation of Gene Ontology categories in..., Maere [/bib_ref]. # Transcription factor analysis The transcription factor analysis was performed using NCA algorithm, and based on reconstructed regulatory networks using RegulonDB [bib_ref] RegulonDB (version 8.0): Omics data sets, evolutionary conservation, regulatory phrases, cross-validated gold..., Salgado [/bib_ref] and the predicted TFA information as previously described [bib_ref] Reconstructing genome-wide regulatory network of E. coli using transcriptome data and predicted..., Fu [/bib_ref]. ## Intracellular ph measurements E. coli MG1655 carrying the pBad24-TorA-GFPmut3* (pJDT1) plasmid [bib_ref] Export of active green fluorescent protein to the periplasm by the twin-arginine..., Thomas [/bib_ref] was grown in 100 mL potassium-modified Luria broth (LBK) [bib_ref] pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia..., Maurer [/bib_ref] media in a 250 mL baffled flask from OD 550 0.05 to midlog (OD 550 ,0.8) with 100 mg/mL ampicillin and 200 mM L-arabinose at 37uC in an orbital incubator shaker at 150 rpm. The cells were harvested at 5,000 g, 4uC for 20 min. and resuspended in phosphate buffered saline (pH = 7.0) diluted to the working concentration (PBS, 10X powder concentrate, Thermo Fisher Scientific, Waltham, MA) without a carbon source to an OD 500 = 0.4. A calibration curve was generated using sodium benzoate, according to [bib_ref] pH of the cytoplasm and periplasm of Escherichia coli: Rapid measurement by..., Wilks [/bib_ref]. E. coli cell suspensions were challenged with C8 or HCl and pH-adjusted to 7.0 before measurement. The intracellular pH was measured by fluorescence For the amino acid supplementation studies, MG1655+pJDT1 was grown overnight in MOPS media as described above. The cells were harvested at 5,000 g, 4uC for 20 min. and resuspended in MOPS with 0.2 mM L-arabinose and incubated at 37uC for 3 h with or without amino acids (arginine and/or glutamate) and C8 at equimolar concentrations of 10 mM. The cells were harvested as treated as above to measure the intracellular pH. For the carboxylic acid production study, E. coli ML103+pXZ18Z+pZS-GFP was grown as described above. The intracellular pH was measured as described above, except the cells were not challenged with exogenous carboxylic acids prior to measurement. c-amino butyric acid measurements MG1655 was grown to midlog (OD 550 ,0.8) as described above with 10 mM C8 and/or 10 mM glutamate added to the growth media. Measurement of c-amino butyric acid was performed according established protocols [bib_ref] A modified rapid enzymatic microtiter plate assay for the quantification of intracellular..., O&apos;byrne [/bib_ref] [bib_ref] Enzyme-based microtiter plate assay for gamma-aminobutyric acid: Application to the screening of..., Tsukatani [/bib_ref]. All chemicals for this assay were purchased from Sigma-Aldrich, St. Louis, MO. Clear UV-transparent microplates were purchased from Corning (Corning, NY). # Membrane lipids analysis The protocol was performed according to [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Briefly, cells were grown to midlog (OD 550 ,0.8) as described above, harvested at 5,000 g, 4uC for 20 min, and then challenged with different concentrations of C8 for 3 h in MOPS pH 7.0 with 2% dextrose. After challenge, the cells were harvested for membrane lipid analysis. ## Proton motive force simulations The total electrochemical force was used to calculate the proton motive force (PMF): [formula] PMF~F DpH zF Dyð1Þ [/formula] and the Nernst potential was used to calculate the contributing forces: [formula] F DpH~2 :3RT zF (pH i {pH e )ð2ÞF Dy~2 :3RT zF log ( ions e ions i )ð3Þ [/formula] where F DpH is the electrochemical force-associated pH-gradient, F Dy is the electrochemical force-associated ion-gradient, R is the gas constant, T is the temperature, z is the molar ion charge, and F is Faraday's constant. Intracellular and extracellular ion abundances are represented by ions i and ions e , respectively. Use of these equations have been previously reviewed [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref] [bib_ref] The measurement of membrane potential and delta-pH in cells, organelles, and vesicles, Rottenberg [/bib_ref]. # Statistical analysis The p-values were obtained using one-way ANOVA and Tukey-Kramer pairs analysis with the JMP v/8.02 statistical program (SAS Institute, Cary, NC, USA). We used a p-value cutoff of 0.05 to determine significance. In the amino acid supplementation survey, we applied the Bonferroni correction factor and adjusted the P-value cutoff to 0.0025. # Results ## Transcriptome analysis highlights the activation of acid resistance systems The information gained by transcriptome analysis is valuable in many applications, especially in the area of tolerance for biorenewable chemicals production. Tolerance is a complex phenotype that covers a diverse space in the bacterial genome [bib_ref] Engineering genomes in multiplex, Woodruff [/bib_ref]. For this reason, transcriptomics is a critical tool that allows for a global measurement of the cellular response network of stressors, such as challenge with carboxylic acids. Therefore, we performed transcriptome analysis of E. coli MG1655 during midlog growth in MOPS minimal media with and without 10 mM octanoic acid, where octanoic acid serves as a representative carboxylic acid. Both cultures had an initial pH of 7.0; 10 mM C8 is sufficient to inhibit growth in this condition by 23% [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. [fig_ref] Table 2: Differentially expressed genes under C8 stress highlight the acid resistance fitness island [/fig_ref] lists all significantly perturbed genes (q,0.05) with at least a 2-fold change in response to C8 challenge. The data is also presented graphically in [fig_ref] Figure 1: Octanoic acid response network [/fig_ref] with log 2 fold changes. Similarly, [fig_ref] Table 3: Transcription factor activities significantly changed [/fig_ref] lists all transcription factors with significantly perturbed (p,0.05) regulatory activity, as determined by network component analysis (NCA). Many of the genes with increased expression in response to C8 challenge are related to acid response, response to and regulation of pH, and biofilm formation. Genes with decreased expression are predominantly attributed to chemotaxis, reduced motility, and flagellum assembly. Several genes associated with membrane function and integrity, such as bhsA and cpxP were also perturbed, where some had increased expression in response to C8 (e.g., cfa) and some had decreased expression (e.g., ompF). These trends are also reflected in the over-represented Gene Ontology (GO) terms, as listed in [fig_ref] Table 1: Strains and plasmids used in this study [/fig_ref]. Similarly, many of the perturbed regulators are also associated with acid response and membrane damage [fig_ref] Figure 1: Octanoic acid response network [/fig_ref]. Most notably, GadE regulates various acid response systems and is primarily controlled at the transcriptional level in response to pH, as previously reviewed [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref]. RcsB is known for both association with capsule biosynthesisand acid resistance [bib_ref] RcsB is required for inducible acid resistance in Escherichia coli and acts..., Johnson [/bib_ref] [bib_ref] RcsB plays a central role in H-NSdependent regulation of motility and acid..., Krin [/bib_ref]. It is known that RcsB activity is regulated by membrane-associated partner proteins in response to a variety of extracellular signalsand is required for maintaining the appropriate cell shape [bib_ref] The Rcs stress response and accessory envelope proteins are required for de..., Ranjit [/bib_ref]. RcsB also regulates motility in an H-NS-dependent manner, which could possibly explain the decrease in motility [bib_ref] RcsB plays a central role in H-NSdependent regulation of motility and acid..., Krin [/bib_ref]. SoxS activation, while not a direct response to acids or membrane integrity, is most likely due to superoxide production as an effect of membrane damage, as previously reviewed [bib_ref] Understanding biocatalyst inhibition by carboxylic acids, Jarboe [/bib_ref]. Activation of Lrp in response to carboxylic acid challenge has been previously described [bib_ref] Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli, Nakanishi [/bib_ref]. Specifically, it was reported that the 4-carbon carboxylic acid butyrate binds to Lrp in a manner that mimics leucine. Therefore, it is possible that a similar interaction occurs with octanoic acid, resulting in activation of Lrp during C8 challenge. The PutA protein shifts between a cytosolic, DNA-binding form and a membrane-associated metabolic enzyme [bib_ref] Regulation of PutA-membrane associations by flavin adenine dinucleotide reduction, Zhang [/bib_ref] [bib_ref] Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression, Commichau [/bib_ref]. The switch to the membrane form is dependent upon the availability of proline and FAD; the membrane-associated form then converts proline into glutamate [bib_ref] Regulation of PutA-membrane associations by flavin adenine dinucleotide reduction, Zhang [/bib_ref]. The abundance of the putA transcript is not changed in our dataset. The observed decrease in PutA TFA therefore suggests that the pool of PutA enzymes shifted from the cytosolic form to the membrane-associated form, indicating a possible increase in proline and/or FAD availability. This potential effect is discussed further below. The putA gene is also transcriptionally activated by the multiple antibiotic resistance regulator MarA, which is also activated in our transcriptome data [bib_ref] Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and..., Pomposiello [/bib_ref]. FruR and MalT are both responsive to metabolites in the early steps of glycolysis. FruR DNA-binding activity is decreased by the metabolites fructose-1-phosphate and fructose-1,6-phosphate [bib_ref] Functioning of a metabolic flux sensor in Escherichia coli, Kochanowski [/bib_ref] ; decreasing FruR activity suggests increased abundance of one or both of these metabolites. MalT is the regulator specific to catabolism of maltose, maltotriose, and starch containing maltose sugars [bib_ref] Network regulation of the Escherichia coli maltose system, Schlegel [/bib_ref]. Several factors may be involved in the activation of MalT. Intriguingly, MalT activity can be stimulated by increased intracellular maltotriose [bib_ref] Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose..., Decker [/bib_ref]. This maltotriose can be produced from unphosphorylated intracellular glucose [bib_ref] Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose..., Decker [/bib_ref]. This unphosphorylated glucose could come from glycogen or possibly enter through the damaged cellular membrane; we have previously quantified this membrane ''leakiness'' using Mg 2+ as a reporter molecule [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. There are other potential sources of unphosphorylated glucose, such as the galactose transporter [bib_ref] Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose..., Decker [/bib_ref]. Two of the most often cited means of carboxylic acid toxicity are intracellular acidification and membrane damage [bib_ref] Perspectives on the use of organic acids and short chain fatty acids..., Ricke [/bib_ref] , and many of the genes and regulators perturbed in our dataset can be attributed to these two effects. Membrane damage in this condition has recently been quantified [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. However, the acidification effect remains relatively undercharacterized. ## Octanoic acid lowers the intracellular ph We used a pH-dependent modified green fluorescent protein [bib_ref] pH of the cytoplasm and periplasm of Escherichia coli: Rapid measurement by..., Wilks [/bib_ref] to verify and quantify the acidification effect of carboxylic acid challenge at neutral pH. We used the familiar example of HCl challenge as a positive control for this system. Addition of 20 mM HCl without readjusting media pH leads to a severe decrease in intracellular pH to 5.32 [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref]. As expected, adjustment of media pH back to neutral after addition of HCl is associated with the appropriate near-neutral intracellular pH. Contrastingly, addition of 10 or 20 mM C8, even with adjustment of media pH to 7.0, results in a significant (p,0.05) and dosedependent drop in intracellular pH [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref]. This data not only confirms the acidification effect of C8 challenge in neutral media, but also quantifies this effect. This acidification is consistent with the known permeability of the cell membrane by the neutral protonated form of octanoic acid. Once this neutral form permeates the cell membrane, it is able to deprotonate inside the cell and thereby decrease the intracellular pH [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Normally, the intracellular pH is slightly basic in order to maintain proper membrane transport properties in a neutral pH environment. E. coli uses this pH gradient as a means of nutrient and waste transport. The pK a of C8 is 4.89 [bib_ref] Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management..., Gagliardi [/bib_ref] ; at pH 7.0; the unprotonated form is dominant in the bulk media. The protonated C8 is able to permeate the cell membrane [bib_ref] External concentration of organic acid anions and pH: key independent variables for..., Carpenter [/bib_ref] [bib_ref] Perspectives on the use of organic acids and short chain fatty acids..., Ricke [/bib_ref] [bib_ref] How fatty acids of different chain length enter and leave cells by..., Kamp [/bib_ref] and release protons into the cell, disrupting the pH gradient. In contrast, HCl is a strong inorganic acid lacking the membrane permeability displayed by the largely hydrophobic carboxylic acid. Thus, HCl is able to pass through the cell membrane only via membrane channels (i.e., ion exchange porins) and proton pumps (e.g., ATPase) [bib_ref] Co-evolution of primordial membranes and membrane proteins, Mulkidjanian [/bib_ref]. Given these differences, it is unclear if the acid resistance strategies thoroughly described in the literature [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref] will also apply to carboxylic acids. These results highlight the challenge of working with carboxylic acids, as opposed to strong inorganic acids. The membrane permeability of these carboxylic acids interferes with the ability to control intracellular pH within the appropriate range of 6.0-7.5 [bib_ref] The effect of food preservatives on pH homeostasis in Escherichia coli, Salmond [/bib_ref]. Traditional fermentation systems enable maintenance of appropriate media pH, but it is the intracellular pH that impacts productivity. Thus, strategies are needed to combat this acidification effect in order to maintain biocatalyst functionality. Native acid resistance systems are ineffective in combatting carboxylic acid stress Bacterial acid resistance systems have been previously characterized [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref]. Specifically there are four known acid resistance systems in E. coli. Two of these, acid resistance system 1 (AR1 -hdeAB operon and hdeD) and acid resistance system 2 (AR2 -gad system) all have significantly increased expression in our C8 challenge dataset. Since AR2 is the better characterized [bib_ref] Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase, Capitani [/bib_ref] [bib_ref] Role of glutamate metabolism in bacterial responses towards acid and other stresses, Feehily [/bib_ref] of the two, we chose this system as the basis for our attempts to enable increased carboxylic acid tolerance. AR2 functions by using glutamate as a sink for excess protons, resulting in production of c-amino butyric acid (GABA) [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref] [bib_ref] Role of glutamate metabolism in bacterial responses towards acid and other stresses, Feehily [/bib_ref]. The fact that the genes encoding this system had increased expression during C8 challenge suggests that E. coli is trying to use this system in our condition. This drive to use the glutamatedependent acid resistance system could explain the observed perturbation of the PutA regulator. AR2 relies on extracellular glutamate for full functionality and thus may have limited effectiveness in our minimal media condition. For this reason, we tested the effectiveness of glutamate supplementation in mitigating both the inhibition of growth and intracellular acidification mediated by octanoic acid. Supplementation with 10, 20 or 30 mM glutamate was not effective in increasing tolerance to 10, 20 or 30 mM C8 at neutral pH respectively (unpublished data). Glutamate supplementation was also not effective at mitigating the acidification effect [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref]. Note that the acidification effect presented in [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref] is more severe than that shown in [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref] ; this is due to the fact that cells queried in [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref] were characterized immediately after C8 addition while cells queried in [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref] were incubated in the presence of C8 for 3 hours, providing the opportunity to use the AR2 system. Note that a pH of 5.5 is the limit of detection of the GFP method and therefore the intracellular pH may actually be less than 5.5. We also attempted to utilize acid resistance system 3 (AR3), which relies on arginine as a proton sink. However, supplemental arginine was also ineffective in combating growth inhibition or acidification [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref]. ## The glutamate-dependent acid resistance system is impaired during carboxylic acid challenge The lack of protection conferred by the arginine-and glutamate-dependent acid resistance systems is surprising, especially considering the fact that the expression of the genes encoding the glutamate-dependent system is increased in our dataset. According to the current understanding of the AR2 system, one glutamate molecule can be used as a sink for one intracellular proton. The arginine-dependent system functions similarly. However, neither of these amino acids was able to provide protection in terms of growth or intracellular acidification [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref]. To gain insight into this surprising result, we further characterized the activity of the AR2 system. The product of the proton-dependent glutamate decarboxylation by GadB, GABA, was measured as a reporter of the activity of the AR2 system [fig_ref] Figure 4: Supplementing glutamate increases GABA export [/fig_ref]. GABA is typically released outside of the cell in order to buffer the intracellular pH to physiological levels (,7.4). This export occurs via the GadC antiport in concert with glutamate import. Note that cells are also capable of transforming GABA into succinate [bib_ref] Role of glutamate metabolism in bacterial responses towards acid and other stresses, Feehily [/bib_ref]. Therefore, both the internal and external GABA abundance values are meaningful. Internal abundance values reflect GadB activity, internal glutamate availability and the relative abundance of excess protons. External GABA abundance reflects functionality of the GadC antiporter and external glutamate availability. We observed that in the control condition, the amounts of intracellular and extracellular GABA are relatively equal. While previous studies have measured GABA abundance in Listeria [bib_ref] Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in..., Karatzas [/bib_ref] , Corynebacterium [bib_ref] Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate..., Takahashi [/bib_ref] , Lactobacillus [bib_ref] Enzyme-based microtiter plate assay for gamma-aminobutyric acid: Application to the screening of..., Tsukatani [/bib_ref] and E. coli [bib_ref] A biological role for prokaryotic ClC chloride channels, Iyer [/bib_ref] , to the best of our knowledge, ours are the first reported measurements for E. coli that document steady-state log-phase intracellular and extracellular GABA concentrations. When supplemental glutamate is added to the control condition, secretion of GABA is increased, though the total amount of GABA remains unchanged relative to the unsupplemented control. This lack of change in the total GABA amount is consistent with the lack of excess protons that need to be removed from the cell interior. We have already described the fact that addition of 10 mM C8 to the media while maintaining a neutral media pH both impedes growth [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref] and severely acidifies the cell interior, with or without provision of 10 mM supplemental glutamate [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref]. For the cells challenged with C8 in the absence of glutamate, there was a decrease in intracellular, extracellular and total GABA. For the cells provided with supplemental glutamate during C8 challenge, there was also a decrease in all types of GABA relative to the unsupplemented control. This lack of perturbation to the GABA pools is surprising, given the known excess of intracellular protons in the +C8 condition. Under appropriate functioning, the GadB/ GadC system should be producing and exporting GABA as a proton sink. However, this system is apparently not functioning during carboxylic acid challenge. This lack of function could possibly be attributed to another well-accepted mechanism of carboxylic acid toxicity: membrane damage. Both GadB and GadC have some degree of membraneassociated function [bib_ref] Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase, Capitani [/bib_ref]. The damage caused to the cell membrane by carboxylic acids can result in impaired function of this system, even when the enzymes and the system precursors, glutamate and excess protons, are abundant. Whereas these two mechanisms were previously thought to be distinct modes of inhibition, to the best of our knowledge, we are the first to link the membrane damage and acidification effect during organic acid challenge. This again highlights the differences in mitigating tolerance to carboxylic acids and strong acids. ## Intracellular acidification is less severe during carboxylic acid production Many of the studies of carboxylic acid tolerance are motivated by a desire to produce carboxylic acids at a high titer [bib_ref] Understanding biocatalyst inhibition by carboxylic acids, Jarboe [/bib_ref]. Note that some studies are instead motivated by the use of carboxylic acids as food preservatives [bib_ref] Medium-chain fatty acids and esters, Kabara [/bib_ref]. The data described above quantifies the intracellular acidification of E. coli during exogenous challenge with octanoic acid. However, this response to exogenous challenge does not necessarily correlate to the physiological effects of carboxylic acid production. Therefore, we quantified the intracellular pH during production of carboxylic acids, using a strain that produces a mixture of C14:0 and C16:0 to a titer of roughly 4 mM in minimal media [fig_ref] Figure 5: Production of carboxylic acids in strain ML103+pXZ18Z+pZS-GFP does not significantly change the... [/fig_ref] [bib_ref] Effect of acetate formation pathway and long chain fatty acid CoA-ligase on..., Li [/bib_ref]. Note that this is the same strain for which we observed decreased membrane integrity as product titers increased [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. While intracellular pH values were observed at or below our reliable detection limit of 5.5 during long-term challenge with octanoic acid [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref] , intracellular pH values during carboxylic acid production were above 6.5 [fig_ref] Figure 5: Production of carboxylic acids in strain ML103+pXZ18Z+pZS-GFP does not significantly change the... [/fig_ref]. E. coli K-12 is generally able to maintain an intracellular pH of 7.660.2 when grown in media with a pH between 6.0 and 7.5, as controlled by the addition of ionic buffering agents [bib_ref] Bacterial strategies to inhabit acidic environments, Kobayashi [/bib_ref]. Outside of this range, growth is still observed at pH values between 4.5 and 9.0 [bib_ref] Bacterial strategies to inhabit acidic environments, Kobayashi [/bib_ref] , though growth is inhibited if intracellular pH falls below 7.1 [bib_ref] The effect of food preservatives on pH homeostasis in Escherichia coli, Salmond [/bib_ref]. It has been noted that E. coli does not grow if the intracellular pH falls below 6.0 [bib_ref] The effect of food preservatives on pH homeostasis in Escherichia coli, Salmond [/bib_ref]. It is also worth noting that similar intracellular pH values were observed in our control strain, in which transcription of the thioesterase responsible for cleaving the elongating fatty acids was not induced. This data suggests that unlike carboxylic acid challenge, carboxylic acid production does not result in intracellular acidification. However, the difference in concentration of carboxylic acids may be the main factor responsible for this difference. Our exogenous challenge studies were performed with 10 or 20 mM C8, but the carboxylic acid production strain does not exceed titers of 5 mM. The lack of intracellular acidification during carboxylic acid production could also be due to metabolic effects, as discussed below. To the best of our knowledge, this is the first quantification of intracellular pH during organic acid production. Cyclopropane fatty acids as a protectant to low pH As described above, membrane damage has been previously cited and quantified as a mechanism of biocatalyst inhibition by carboxylic acids [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref] [bib_ref] Membrane stresses induced by overproduction of free fatty acids in Escherichia coli, Lennen [/bib_ref]. Genes and regulators related to membrane damage were perturbed in our transcriptome data. We have also proposed that membrane damage is the reason for poor functioning of the glutamate-dependent acid resistance system. Previous engineering efforts for increasing carboxylic acid tolerance in E. coli have focused on increasing the relative content of saturated lipids in the cell membrane [bib_ref] Modulating membrane composition alters free fatty acid tolerance in Escherichia coli, Lennen [/bib_ref]. These efforts were successful in increasing tolerance to carboxylic acids, but not increasing carboxylic acid production. Given that increased membrane permeability (leakage) is a likely mechanism of carboxylic acid toxicity, it is appealing to implement genetic modifications that increase membrane integrity. Cyclopropane fatty acids are known to have increased bulk in the cell membrane relative to saturated fatty acids [bib_ref] Membrane lipid homeostasis in bacteria, Zhang [/bib_ref]. Additionally, cyclopropane fatty acids have been well-documented in the role in conferring resistance to acidic environments [bib_ref] Membrane cyclopropane fatty acid content is a major factor in acid resistance..., Chang [/bib_ref]. Considering the fact that E. coli inhibition by carboxylic acids is associated with both membrane damage and induction of the acid response, we proposed that increasing the abundance of these cyclopropane fatty acids in the cell membrane could serve as a method of increasing carboxylic acid tolerance. The role of cyclopropane fatty acids in carboxylic acid tolerance was investigated using mutants either completely deficient in cyclopropane fatty acid production (Dcfa) or engineered for increased expression of the Cfa enzyme (cfa++). Note that Cfa is responsible for the S-adenosyl-L-methionine (SAM)-dependent methylation of unsaturated membrane lipids [bib_ref] Escherichia coli cyclopropane fatty acid synthase: Is a bound bicarbonate ion the..., Courtois [/bib_ref]. No cyclopropane fatty acids were observed in our Dcfa mutant [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. Strains with increased cfa expression contained more than 32 mol% cyclopropane fatty acids while less than 11 mol% were observed in the wildtype strain in the control condition [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. The complete lipid profiles are shown in [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref]. Despite this substantial change in cyclopropane fatty acid content in the cell membrane, there was not an observed increase in C8 tolerance [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref] inset) and no change in leakage or fluidity (unpublished data). In fact, strains with the highest cyclopropane fatty acid content actually showed significantly (p,0.05) increased sensitivity to C8 [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. Despite the lack of impact on carboxylic acid tolerance, the data obtained in these experiments provides interesting trends in control of membrane composition. We observed previously that when challenged with C8, E. coli undergoes a dose-dependent, statistically significant decrease in the molar ratio of saturated (i.e,. C16:0) and unsaturated (i.e. C16:1 and C17cyc) membrane lipids (S:U ratio) [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Cells deficient in cyclopropane fatty acid production have a saturated:unsaturated ratio in the control condition that is significantly lower than the wildtype strain [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. However, as the C8 concentration increases, the S:U ratio in the Dcfa strain approaches the S:U ratio observed in the wildtype strain. Contrastingly, cells with increased cyclopropane fatty acid content (cfa++) show a similar S:U ratio as the wildtype strain in the control condition, but when C8 is added at increasing concentration, the observed S:U ratio is increased more than 2fold relative to the wildtype [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. Similar patterns were observed during growth sensitivity measurements. As the C8 concentration increases, the Dcfa strain shows growth rates increasingly similar to the wildtype strain, while the cfa++ strain diverges from the wildtype [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. Another metric for assessing membrane composition is average lipid length. We previously reported that during growth in the presence of C8, the average lipid length increased in a significant, dose-dependent manner [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Cells with increased expression of cfa are able to maintain the same average lipid length as the wildtype in the control condition, but in the presence of C8 the lipid length decreases [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. This decrease in average lipid length represents a shift from lipids with an 18-carbon chain (C18:0, C18:1, C19cyc) to lipids with a 16-carbon chain (C16:0, C16:1 and C17cyc). Contrastingly, the Dcfa strain followed the wildtype trend by significantly increasing the average lipid length. This was accomplished by decreasing 16:1 and increasing C18:1 [fig_ref] Figure 6: cfa mutants with altered the membrane lipid profile, S [/fig_ref]. Thus, separation of cfa expression from the wildtype control circuits impacts not only the saturated:unsaturated fatty acid ratio, but also the average lipid length. This highlights the potential importance of Cfa and cyclopropane fatty acids in maintaining appropriate membrane properties. ## Interaction of lrp and octanoic acid does not contribute to c8-mediated growth inhibition It has been previously noted that small carboxylic acids can interact with the global regulator Lrp in a manner than mimics its standard cofactor, leucine [bib_ref] Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli, Nakanishi [/bib_ref]. Thus, it is possible that the observed activation of Lrp in our dataset was caused by interaction of Lrp and octanoic acid. Lrp is a global regulator, primarily of amino acid biosynthesis [bib_ref] The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli, Calvo [/bib_ref] and its activation by a false signal, such as octanoate, could result in inappropriate repression of biosynthesis-associated genes, resulting in growth inhibition. We tested the octanoic acid tolerance of both an lrp deletion mutant and a strain engineered for increased expression of lrp. However, consistent with previous reports [bib_ref] The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli, Calvo [/bib_ref] , the deletion mutant grew poorly in our minimal media growth condition and data regarding carboxylic acid sensitivity was ambiguous [fig_ref] Figure 7: lrp mutations do not improve E [/fig_ref]. Specifically, the overall growth rate was decreased relative to the wildtype at all C8 concentrations tested, but the relative sensitivity to C8 did not appear to be altered. Similar trends were observed for the strain engineered for increased lrp expression [fig_ref] Figure 7: lrp mutations do not improve E [/fig_ref]. We proposed that this difficulty in establishing the role of the potential C8-Lrp interaction in C8 tolerance is due to the global regulatory role of Lrp. If C8 is truly serving as a false signal for controlling Lrp activity, this is most likely to impact growth in the form of inappropriate repression of amino acid biosynthesis. Therefore, we tested each of the individual amino acids, as well as a mixture of biosynthetic building blocks in the form of casamino acids, for their ability to confer tolerance to C8. If one or more of these amino acids is limiting during growth in the presence of C8, supplementation with these amino acids should increase C8 tolerance. However, none of the individual amino acid supplements or supplemental casamino acids enabled significantly increased growth in the presence of 10 mM C8 relative to the unsupplemented condition (unpublished data). It should also be noted that none of these supplements significantly inhibited growth in the non-C8 condition. The cell can initiate a response that gives times to adjust the PMF to the target range. Assumption 4: Rapid addition of a strong mineral acid (e.g., HCl) adds an equal amount of anions and protons, which does not change the membrane potential. Assumption 5: C8 is at a sufficiently high concentration that it rapidly integrates into the cell membrane and releases its proton inside the cell, which is not reversible. A negative (2) PMF indicates protons diffusing into the cell, whereas a positive (+) PMF indicates protons diffusing out of the cell. A negative (2)Dy indicates an overall negative ion gradient due to anions, whereas a positive (+)Dy indicates an overall positive ion gradient due to cations. The mol ions is the molar ratio from the normal condition. pH i -intracellular pH; pH e -extracellular pH; F DpH -DpH associated electrochemical force; F Dy -membrane potential associated electrochemical force; PMF-proton motive force; A 2 (red) C8 anion; A 2 (green) another anion; C+ any cation. doi:10.1371/journal.pone.0089580.g008 Therefore, even if there is a significant interaction between octanoic acid and Lrp, this interaction does not appear to be contributing to C8-mediated growth inhibition in our condition. Octanoic acid causes inversion of the pH gradient leading to anion accumulation in order to maintain the proton motive force While it does not appear at this point that intracellular acidification is a significant challenge for carboxylic acid production, that perspective may change as we attain higher carboxylic acid titers. Given the qualitative differences observed between challenge with strong inorganic acids and carboxylic acids, we have applied a quantitative modeling approach to identifying additional differences between these two types of acids. The proton motive force (PMF, typically expressed in mV) is a function of both the difference in intracellular and extracellular pH values (DpH) and differences in internal and external electrochemical charge (membrane potential, Dy) as shown above in equation [bib_ref] Manufacturing molecules through metabolic engineering, Keasling [/bib_ref]. [fig_ref] Figure 8: Simulations of the effect of the electrochemical gradients on the proton motive... [/fig_ref] shows our simulations of the pH gradients, membrane potential, and the resultant PMF under different environmental conditions. When cells are shocked with an inorganic acid, such as HCl, the PMF becomes larger than normal and protons initially overwhelm the cell interior. But after a period of adaptation, accumulation of cations can enable reversion of the PMF back to the physiological range, even though the intracellular pH in the adapted state is decreased relative to the original intracellular pH. Such cation accumulation, in the form of potassium, has been previously described for E. coli K-12 [bib_ref] The ability of Escherichia coli O157:H7 to decrease its intracellular pH and..., Diez-Gonzalez [/bib_ref]. This cation accumulation will lead to a change in the transmembrane potential (Dy) that contributes to stabilization of the PMF. This stabilization of the PMF creates hyperpolarization of the membrane, as evidenced by very large DpH and Dy values. This hyperpolarization can be mediated by the release of chloride anions through voltage-gated channels as discussed in the literature [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref] [bib_ref] A biological role for prokaryotic ClC chloride channels, Iyer [/bib_ref] [bib_ref] Escherichia coli glutamate-and arginine-dependent acid resistance systems increase internal pH and reverse..., Richard [/bib_ref] [bib_ref] Secondary active transport mediated by a prokaryotic homologue of ClC Cl-channels, Accardi [/bib_ref]. This type of change in Dy from negative to positive during challenge with inorganic acids has been previously described [bib_ref] Escherichia coli glutamate-and arginine-dependent acid resistance systems increase internal pH and reverse..., Richard [/bib_ref]. Given the ability of our model to accurately describe the E. coli response to inorganic acids, here we have applied this model to carboxylic acid challenge. When cells are shocked with carboxylic acids while maintaining a neutral extracellular pH, the DpH value is actually inverted. This inversion of DpH results in a transient inversion in PMF. In order to return to the viable PMF range and appropriate directionality, cells must accumulate intracellular anions, as opposed to the cations accumulated after challenge with inorganic acids. Our model predicts that this anion accumulation will increase the intracellular anion pool by 3-fold. This anion accumulation in response to carboxylic acid challenge, as opposed to the cation accumulation seen with inorganic acids, is consistent with literature reports. For example, when E. coli O157:H7 was treated with sufficient external acetate to drop the intracellular pH to below 6.5 while maintaining a constant media pH of 5.9, the amount of intracellular acetate increased 3-fold while intracellular potassium levels remained relatively constant [bib_ref] The ability of Escherichia coli O157:H7 to decrease its intracellular pH and..., Diez-Gonzalez [/bib_ref]. Thus, physiological changes that enable maintenance of the appropriate PMF differ between carboxylic acids and inorganic acids. Specifically, our model predicts that challenge with carboxylic acids will result in a transient inversion of the proton motive force. This prediction is a key component of our analysis, and while it is supported by literature data [bib_ref] The ability of Escherichia coli O157:H7 to decrease its intracellular pH and..., Diez-Gonzalez [/bib_ref] , the existence of this transient inversion is not widely known. Note that E. coli K-12 normally maintains a PMF in the range of 2140 to 2180 mV [bib_ref] Escherichia coli acid resistance: Tales of an amateur acidophile, Foster [/bib_ref]. # Discussion While transcriptome analysis is informative about the cellular response network [fig_ref] Figure 1: Octanoic acid response network [/fig_ref] and [fig_ref] Table 2: Differentially expressed genes under C8 stress highlight the acid resistance fitness island [/fig_ref] during challenge with an inhibitor such as octanoic acid, here we have supported these findings with other data that quantitatively describe the physiological state of the cell. We have confirmed that octanoic acid acidifies the cytosol of the E. coli cell [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref]. Our analysis predicts that this acidification inverts the proton gradient and substantially changes the PMF [fig_ref] Figure 8: Simulations of the effect of the electrochemical gradients on the proton motive... [/fig_ref]. Such a change alters the physiological state of the cell in a way that is fundamentally different from inorganic acids such as HCl. Whereas adaptation to HCl includes accumulation of intracellular cations, organic acid stress adaptation includes accumulation of anions in order to revert the PMF to the appropriate range and directionality. Our simulations could be confirmed in future experiments by measuring the intracellular pH, extracellular pH, the transmembrane potential, and intracellular ion concentrations. We have previously described the negative effects of octanoic acid on the cellular membrane of E. coli [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Here we have further explored the effect of octanoic acid on membrane proteins such as those associated with acid resistance [fig_ref] Figure 4: Supplementing glutamate increases GABA export [/fig_ref]. While supplementing E. coli with glutamate aids in export of the decarboxylation product GABA, it does not support the glutamate-dependent acid resistance system under octanoic acid stress [fig_ref] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification [/fig_ref] and [fig_ref] Figure 4: Supplementing glutamate increases GABA export [/fig_ref]. We proposed that this system is defective during octanoic acid challenge due to membrane damage. It is known that cyclopropane fatty acids aid in tolerance of acid stress, presumably by slowing the transport of protons into the cell [bib_ref] Membrane cyclopropane fatty acid content is a major factor in acid resistance..., Chang [/bib_ref] [bib_ref] Escherichia coli cyclopropane fatty acid synthase: Is a bound bicarbonate ion the..., Courtois [/bib_ref]. Here we have noted that Cfa is involved with complex control of membrane properties, including the saturated:unsaturated lipid ratio and the average lipid length. The effect of Cfa on membrane properties is reflected by the specific growth rate. Our efforts to engineer the membrane in order to confer carboxylic acid tolerance suggests that precise control of the membrane is required for sufficient growth, similar to the conclusions of other researchers [bib_ref] Modulating membrane composition alters free fatty acid tolerance in Escherichia coli, Lennen [/bib_ref]. It was reported previously that both exogenous addition and intracellular production of carboxylic acids causes disruption of the membrane [bib_ref] The damaging effects of short chain fatty acids on Escherichia coli membranes, Royce [/bib_ref]. Here we report the differences in how carboxylic acids affect the intracellular pH. While exogenous addition of carboxylic acids causes a significant drop in the intracellular pH, intracellular production of carboxylic acids does not. However, the production strains achieve a much lower titer than the concentrations used in our exogenous challenge studies. It may be that the acidification effect may not become significant in carboxylic acid production strains until higher product titers are achieved. Another potentially-important aspect of carboxylic acid production relates to the distribution of these acids between the protonated and anionic forms within the cell interior. The protonated carboxylic acids can more easily diffuse through the cell membrane than the anionic form [bib_ref] Effect of the alkyl chain length of monocarboxylic acid on the permeation..., Evtodienko [/bib_ref]. A buildup of intracellular anionic carboxylic acids will shift the equilibrium to the protonated form. The neutral lipophilic fatty acids will then diffuse through the membrane, where it can disassociate, assuming that the media pH is greater than the pK a of the fatty acid. # Conclusions These results highlight the ability of transcriptome analysis to guide quantitative experiments in establishing the mechanisms of toxicity of various inhibitory compounds. These mechanisms of toxicity can differ depending on whether the inhibitor is provided exogenously or produced by the cell. Thus, testing of proposed hypotheses in engineered strains is helpful in prioritizing engineering strategies. At this point, it still appears that membrane damage is the most pressing issue limiting carboxylic acid production. Strategies that enable increased membrane integrity may enable increased carboxylic acid production titers, so that intracellular acidification becomes problematic. [fig_ref] Figure 1: Octanoic acid response network [/fig_ref] Transcriptome data of significantly perturbed genes during E. coli challenged with octanoic acid. The criteria for significantly perturbed genes: q,0.05 with a log 2 ratio ,21.0 or .1.0. Positive values are an increase in abundance and negative values are a decrease in abundance of transcripts. MG1655 was challenged with 10 mM octanoic acid in MOPS with 2% dextrose at 37uC, 150 rpm. (TIF) [fig_ref] Figure 2: Octanoic acid challenge decreases the intracellular pH [/fig_ref] The complete lipid profiles of E. coli strains with engineered cyclopropane fatty acid content. The complete membrane lipid profile of MG1655 and strains with altered cfa expression. Strains were incubated with 0-30 mM C8, pH = 7.0. ## Supporting information C12:0-lauric acid, C14:0-myristic acid, C16:0-palmitic acid, C16:1-palmitoleic acid, C17cyc-cyclopropane C17:0, C18:1vaccenic acid, C18:0-stearic acid, C19cyc-cyclopropane C19:0. (TIF) Table S1 Over-represented Gene Ontology terms.Over-represented GO terms (DOCX) [fig] Figure 1: Octanoic acid response network. MG1655 was challenged with sufficient octanoic acid to inhibit growth by 23% (10 mM) in MOPS minimal media at pH 7.0, 37uC, 150 rpm. This diagram shows central metabolism and highlights the regulatory effect of regulators with significantly perturbed activity during C8 challenge, as identified by network component analysis. Dashed lines indicate regulatory connections that were proposed in our previous analysis[30]. Mechanisms for changes in transcription factor activity are discussed in the text. doi:10.1371/journal.pone.0089580.g001 [/fig] [fig] Figure 2: Octanoic acid challenge decreases the intracellular pH. E. coli MG1655 pJTD1 was grown to midlog in minimal media at pH 7.0 and resuspended in media containing C8 or hydrochloric acid (HCl). Values are the average of 4 biological replicates, with error bars indicating the standard deviation. doi:10.1371/journal.pone.0089580.g002 [/fig] [fig] Figure 3: Supplementation with arginine or glutamate does not mitigate intracellular acidification. Measurements of the intracellular pH of E. coli MG1655 pJTD1 during C8 challenge while grown in the presence of supplemental arginine and glutamate. The cells were incubated for 3 h at 37uC in MOPS media to allow utilization of the amino acid-dependent acid resistance systems. All concentrations are 10 mM. doi:10.1371/journal.pone.0089580.g003 [/fig] [fig] Figure 4: Supplementing glutamate increases GABA export; however, addition of C8 reduces GABA accumulation and export. GABA measurements of MG1655 during log phase growth in MOPS with 2% dextrose at 37uC, 150 rpm. All concentrations are 10 mM. GABA: c-amino butyric acid. doi:10.1371/journal.pone.0089580.g004 [/fig] [fig] Figure 5: Production of carboxylic acids in strain ML103+pXZ18Z+pZS-GFP does not significantly change the intracellular pH. Shake flasks of E. coli producing predominately C14:0 and C16:0 carboxylic acids in M9 media with 1.5% dextrose at 30uC. IPTG induces the pXZ18Z plasmid carrying a thioesterase and a b-hydroxyacyl-ACP dehydratase. The intracellular pH values are the average of four biological replicates and four technical replicates. The error bars indicate the standard deviation. doi:10.1371/journal.pone.0089580.g005 [/fig] [fig] Figure 6: cfa mutants with altered the membrane lipid profile, S:U ratio and the average lipid length. a: Membrane lipid profile of MG1655 and strains with altered cfa expression. Strains were incubated with 0-30 mM C8, pH = 7.0. Inset: specific growth rate in the log phase of E. coli with varying cfa expression. C16:0-palmitic acid, C16:1-palmitoleic acid, C17cyc-cyclopropane C17:0, C18:1-vaccenic acid, C18:0-stearic acid, C19cyc-cyclopropane C19:0. The complete lipid profiles are shown inFigure S2of the Supporting Information. Membrane properties are calculated from a to obtain: b: saturated:unsaturated lipid ratio and c: average lipid length. doi:10.1371/journal.pone.0089580.g006 [/fig] [fig] Figure 7: lrp mutations do not improve E. coli growth rate upon addition of octanoic acid. The specific growth rate of strains with altered lrp expression during the log phase in MOPS with 2% dextrose at 37uC, 150 rpm. doi:10.1371/journal.pone.0089580.g007 [/fig] [fig] Figure 8: Simulations of the effect of the electrochemical gradients on the proton motive force. The appropriate range for E. coli cells is 2140 to 2180 mV. pH values are measurements from Figure 2 at 20 mM. Assumption 1: An average of 2160 mV is used for the normal PMF. The target PMF is within the range of 2140 to 2180 mV. Assumption 2: Shock conditions are the instantaneous outcome of the PMF after an extreme change in the environment. Assumption 3: [/fig] [table] Table 1: Strains and plasmids used in this study. [/table] [table] Table 2: Differentially expressed genes under C8 stress highlight the acid resistance fitness island. [/table] [table] Table 3: Transcription factor activities significantly changed. [/table]

Emotional intelligence of nurses caring for COVID-19 patients: A cross-sectional study # Introduction World health organization declared the COVID-19 crisis as a pandemic with a public health emergency [bib_ref] COVID-19: Emerging compassion, courage and resilience in the face of misinformation and..., Smith [/bib_ref]. Health care providers have a high tendency to be infected with the virus. The prevalence of COVID-19 in health care workers (HCW) is more than the general population. HCWs are prone to psychological problems . Studies report that this pandemic could increase the risk of psychological symptoms in asymptomatic populations and exacerbate the symptoms in previously diagnosed psychological problems [bib_ref] Keeping positive and building strength: The role of affect and team leadership..., Sommer [/bib_ref]. Anger, confusion, and post-traumatic stress disorder are reported as a consequence of this pandemic [bib_ref] Work stress among Chinese nurses to support Wuhan in fighting against COVID-19..., Mo [/bib_ref]. HCWs who are working in the frontline are at risk for psychological disorders and sometimes the prevalence of disorders such as depression and anxiety in HCWs is reported more than the general population [bib_ref] Exploring the relationship between perceived emotional intelligence, coping, social support and mental..., Montes-Berges [/bib_ref]. As a result, we should take measures to improve the psychological health of frontline nurses to prevent further damage to the nurses and their patients [bib_ref] The relationship between emotional intelligence with job and individual characteristics of nursing..., Doherty [/bib_ref]. Emotional intelligence (EI) is one of the most important aspects that improve psychological health. EI is the ability to recognize, understand, and regulate emotions and use them in life. Also, EI is recognized as a core variable affecting job performance [bib_ref] The impact of emotional intelligence in health care professionals on caring behaviour..., Nightingale [/bib_ref]. It is also a key factor for Creating, reinforcing the emotional capacity that nurses need to interact with patients, and bearing the emotional burden. The level of EI may vary between different occupations, especially among health care professionals [bib_ref] Emotional intelligence of nursing applicants and factors related to it: A cross-sectional..., Talman [/bib_ref]. Some studies report a strong correlation between EI and nurses' performance. They also report the EI of nurses high [bib_ref] The impact of emotional intelligence on job performance during COVID-19 crisis: A..., Alonazi [/bib_ref]. The intensifying of the COVID-19 pandemic plays a strong role in emotional and psychological disorders between HCWs especially nurses [bib_ref] Psychological stress of medical staffs during outbreak of COVID-19 and adjustment strategy, Wu [/bib_ref]. The level of emotional intelligence during the COVID-19 crisis has been evaluated in previous studies [bib_ref] The impact of emotional intelligence on job performance during COVID-19 crisis: A..., Alonazi [/bib_ref] [bib_ref] Navigating COVID-19 with emotional intelligence, Baba [/bib_ref]. Improving emotional intelligence skills affects nurses' performance [bib_ref] The impact of emotional intelligence on job performance during COVID-19 crisis: A..., Alonazi [/bib_ref] and less experiencing negative emotions. This study was to assess the EI in nurses caring for COVID-19 patients. The results of present study besides having theoretical implications could open pathways for conducting more research. # Methods ## Aim This descriptive cross-sectional study was done from May to July 2020 in Tehran, Iran. The aim of the study was to assess the EI of nurses caring for COVID-19 patients. ## Sample All nurses who were working in COVID-19 wards were invited to participate in the study. Those with at least an associate degree in nursing who were caring for COVID-19 patients and had willing to participate in the study were included. Totally 211 nurses participated in the study. ## Data collection Sampling was done using a web-based online questionnaire. The questionnaire was sent online to 250 people via email and social media. The required time for responding the questionnaire was 5 to 7 min. If no response was given, after one week a reminder was sent. Finally, 211 questionnaires were completed. These questionnaires were assessed and analyzed using statistical software. Totally the response rate was reported 87.2%. The questionnaire had two parts. The first section was demographic and job description and the second part was the Emotional Intelligence questionnaire developed by Brad berry and Greaves in 2005. All nurses included in the study worked in the wards where COVID-19 patients were admitted. Nurses from both private and governmental sectors were included. Also, they were working in both general and intensive care wards. Participants were informed about the study objective and the confidentiality of the data. They had the right to withdraw the study based on their desire. The informed written consent was taken from all participants. This questionnaire has 28 items and four aspects of Self-awareness (questions 1-6), self-management (questions 7-15), social awareness (questions 16-21), and relationship management (questions 22-28). The scoring of this questionnaire is based on a Likert scale from 1 to 6. The sum of the score is the total score of each aspect. Then we computed them and calculate the total score of the questionnaire. For better understanding, the total score of the questionnaire was computed to 0 to 100. Having a higher score means higher EI. This questionnaire is translated and transcultural adapted in Persian. The reliability of this test in studies based on internal consistency method, Cronbach's alpha coefficient has been reported 0.83. The validity of this scale has been determined by a correlation coefficient of r = 0.67 and P < 0.01 [bib_ref] Bradberry-Greaves' emotional intelligence test: Preliminary norming-process, Ganji [/bib_ref]. # Ethical consideration The present study was approved by the Ethics Committee of Baqiyatallah University of Medical Sciences, Tehran, Iran, with code IR. BMSU.REC.1399.227. # Statistical analysis Data were analyzed using the Statistical Package for the Social Sciences (SPSS) 25.0 statistical package (Chicago, IL, USA), and a two-sided P-value > 0.05 was considered as a statistically significant difference. Categorical variables are presented as frequency rates and percentages, and continuous variables are described using mean ± standard deviation (SD) values. Data analysis was performed using descriptive and inferential statistics (independent t-test, Multinomial logistic regression, Spearman's rho correlation coefficient). The normality of numeric variables was tested using the Kolmogorov-Smirnov test. The comparison of demographic characteristics and job experience parameters was done by independent t-test for continuous variables and Chi-square test or Fisher's exact test for categorical variables. # Results Totally 211 nurses participated in the study. Of these, 52.7% of them were female, 71.6% were married, and 72.5% of them were BSc. The mean age and work experience of participants were 34 (7.85), and 9.76 (6.67), respectively. Most of the nurses were working in critical care wards and caring for critical patients. Other demographic data are presented in [fig_ref] Table 1: General information of respondents [/fig_ref]. Mean of EI of nurses was 63.19 (8.22) and in different subscales of the EI self-awareness had the highest grade and self-management had the lowest grade. Details are summarized in. Independent t-test did not show a statistically significant difference in the mean scores of emotional intelligence in terms of gender and type of ward. Also, the independent t-test showed that married nurses had higher EI scores compared to single nurses. The one-way ANOVA statistical test didn't show any significant difference in the scores of EI based on caring level, education, and employment status . Married nurses had a higher score in subscales of self-management and relationship management compared to singles. Also, nurses who were caring for more complex patients had a higher score in the relationship management subscale . # Discussion Based on available data the EI of nurses caring for COVID-19 patients is not well studied. This study was done to assess the EI of nurses caring for COVID-19 patients. Nurses had a moderate score in EI. The EI of married nurses was higher than single. In general, the highest and lowest scores were for self-awareness and self-management, respectively. Different scores are reported for EI in different studies. Some studies report that the EI of nurses is optimal [bib_ref] Measuring emotional intelligence of nursing student, Barkhordari [/bib_ref] [bib_ref] Emotional intelligence and its impact on the emotional factors among nurses, Konstantinou [/bib_ref] [bib_ref] The relationship between emotional intelligence and coping styles of nurses in hospitals..., Rostami [/bib_ref] [bib_ref] On the relationship between emotional intelligence and demographical variables in nurses, Saeid [/bib_ref]. In some studies nurses' EI score is reported low [bib_ref] Measuring the emotional intelligence of clinical staff nurses: An approach for improving..., Codier [/bib_ref] [bib_ref] The emotional intelligence profile of successful staff nurses, Harper [/bib_ref]. The reason for this discrepancy could be the assessment tool or the definition of EI. Despite the difference in assessment tools, the results of these studies affirm that the EI of nurses needs more considerations and some measures should be planned. The nature of the nursing profession and the diversity of situations they face require the nurses have high EI [bib_ref] Measuring emotional intelligence of nursing student, Barkhordari [/bib_ref]. The EI, during Most of nurses participated in this study were Female, married, and were working in ICU wards. situations such as the COVID-19 outbreak, could have a protective role for negative emotions such as fear, anxiety, and sadness [bib_ref] Trait emotional intelligence and emotional experiences during the COVID-19 pandemic outbreak in..., Moroń [/bib_ref]. This issue is more important due to the effects of a pandemic on the occurrence of psychological symptoms and the mental health of health care workers (Di [bib_ref] Mental health of healthcare workers during the COVID-19 pandemic in Italy, Tella [/bib_ref] [bib_ref] Nurses' mental health during the Covid-19 outbreak: A cross-sectional study, Sampaio [/bib_ref]. One of the main challenges managers are faced is the psychological well-being of health care workers [bib_ref] Managing mental health challenges faced by healthcare workers during covid-19 pandemic, Greenberg [/bib_ref] [bib_ref] Covid-19: Supporting nurses' psychological and mental health, Maben [/bib_ref]. This issue could have multiple negative consequences such as job dissatisfaction, stress, burnout, reduced performance, and the quality of care. EI gives the nurses the power to think better during critical and difficult situations and make better decisions by controlling the psychological reactions [bib_ref] Emotional intelligence in nursing, models and methods of measurement, Kelishami [/bib_ref]. Also, EI is an effective strategy for reducing job stress and promoting resiliency [bib_ref] Promoting emotional intelligence and resilience in undergraduate nursing students: An integrative review, Cleary [/bib_ref] [bib_ref] The effect of Emotional Intelligence (EI) training on nurses' resiliency in Department..., Khoshnazary [/bib_ref]. In terms of clinical performance, improving EI will lead to improving the quality of nursing care [bib_ref] The relationships in emotional intelligence, job satisfaction, and quality of nursing service..., Jang [/bib_ref]. Self-awareness and self-management had the highest and lowest scores, respectively. This means that nurses have a high competency in recognizing their emotions, but their ability to manage them is not desirable. The results of similar studies also confirm the findings of the present study [bib_ref] The relationships among emotional intelligence, interpersonal relationship, and job satisfaction of clinical..., Ko [/bib_ref] [bib_ref] The relationship of emotional intelligence and its dimensions on communication skills among..., Mosadegh Rad [/bib_ref] [bib_ref] Emotional intelligence among nursing students: Findings from a crosssectional study, Š Tiglic [/bib_ref]. Trying to improve the self-management is necessary. Selfmanagement causes better performance in different stressful situations such as discordant with colleagues, denial, and emotions of patients and family members [bib_ref] The essentials: Using emotional intelligence to curtail bullying in the workplace, Meires [/bib_ref]. Considering the dimensions of EI is very important. Because we can assess the nurse and diagnose the area the person is weak and plan for improving it. Self-awareness and self-management are considered as individual capabilities of emotional intelligence and social awareness and relationship management as social capabilities [bib_ref] Bradberry-Greaves' emotional intelligence test: Preliminary norming-process, Ganji [/bib_ref]. Therefore, we should not focus only on the overall score of emotional intelligence, but the score of dimensions are more important. Married nurses had higher scores in EI. Also, the score of selfmanagement and relationship management were higher in married nurses. This difference also has been mentioned in other studies [bib_ref] The relationship between emotional intelligence with job and individual characteristics of nursing..., Doherty [/bib_ref]. This can be due to the involvement of married people in issues such as personal disputes and unfulfilled expectations that require empathy and adjustment. Therefore, it can be an exercise to improve and develop emotional intelligence. There is no significant relationship between gender and age with emotional intelligence in other similar studies [bib_ref] Application of structural equations modeling to assess relationship among emotional intelligence, general..., Khandan [/bib_ref] [bib_ref] Identifying emotional intelligence in professional nursing practice, Kooker [/bib_ref] [bib_ref] The role of emotional intelligence in building interpersonal communication skills, Petrovici [/bib_ref]. However, differences in the type of tools used to assess emotional intelligence can also be particularly effective. In the present study, there was no difference between the EI of nurses caring for patients with COVID19 in the normal and critical wards. In terms of dimensions of emotional intelligence, nurses caring for clinically stable COVID19 patients had a higher relationship management score. In the literature review, the results indicate a moderate and low level of emotional intelligence in nurses working in the intensive care unit [bib_ref] Assessment and comparison of emotional intelligence of nurses in general and intensive..., Saeed [/bib_ref]. However, COVID19 pandemic affects the mental health of health Totally, nurses' emotional intelligence was moderate. Also, selfawareness and self-management had highest and lowest scores, respectively. ## Table 3 The relationship between emotional intelligence and Dimensions with demographic characteristics. care workers and can affect the quality of care provided. Therefore, it is necessary to pay attention to the emotional intelligence of nurses caring for critically ill patients with COVID19. # Limitations Due to limited access to research samples, questionnaires were sent and completed online. So we could not comply fully with our sampling schedule and plan. # Conclusion The EI of nurses caring for patients with COVID19 was moderate. In terms of dimensions, the highest score was related to individual abilities (self-awareness and self-management). According to the results of the present study, the development of emotional intelligence based on the characteristics of nurses is very important in recognizing and managing their emotions. Due to the continuation of this pandemic and the possibility of mental and physical fatigue of health care workers, improving emotional intelligence can be effective in resilience and stability of the psychological status of employees. ## Declaration of competing interest We declare that we have no conflicts of interest. [table] Table 1: General information of respondents. [/table]

Disseminated tumour cells as a prognostic biomarker in colorectal cancer BACKGROUND: The study was performed to determine detection rate and prognostic relevance of disseminated tumour cells (DTC) in patients receiving curatively intended surgery for colorectal cancer (CRC). METHODS: The study population consisted of 235 patients with CRC prospectively recruited from five hospitals in the Oslo region. Bone marrow (BM) aspirates were collected at the time of surgery and the presence of DTC was determined by two immunological methods; immunomagnetic selection (using an anti-EpCAM antibody) and immunocytochemistry (using a pan-cytokeratin antibody). Associations between the presence of DTC and metastasis-free, disease-specific and overall survival were analysed using univariate and multivariate methods. RESULTS: Disseminated tumour cells were detected in 41 (17%) and 28 (12%) of the 235 examined BM samples by immunomagnetic selection and immunocytochemistry, respectively, with only five samples being positive with both methods. The presence of DTC was associated with adverse outcome (metastasis-free, disease-specific and overall survival) in univariate and multivariate analyses. CONCLUSION: The presence of DTC was associated with adverse prognosis in this cohort of patients curatively resected for CRC, suggesting that DTC detection still holds promise as a biomarker in CRC. In colorectal cancer (CRC), treatment decisions are still made almost exclusively based on clinicopathological parameters as described by Dukes almost a century ago [bib_ref] The classification of cancer of the rectum, Dukes [/bib_ref] , and the search for prognostic biomarkers to improve patient stratification for adjuvant treatment and intensified postoperative surveillance is highly warranted. Despite advances in diagnosis and treatment, a significant proportion (up to 50%) of curatively resected patients develops disease recurrence, primarily as liver and lung metastases [bib_ref] Colon cancer survival rates with the New American Joint Committee on cancer..., O&apos;connell [/bib_ref] [bib_ref] Surveillance strategies after curative treatment of colorectal cancer, Pfister [/bib_ref]. Metastasis development in patients without discernable metastatic disease at the time of primary surgery reflects preceding dissemination of tumour cells with metastatic properties to target organs. Over the last couple of decades, the identification of tumour cells in blood and bone marrow (BM) has been proposed as a potential biomarker of adverse prognosis in solid tumours [bib_ref] Cancer micrometastases, Pantel [/bib_ref]. Analyses of tumour cells derived from blood and BM suggest that micrometastases represent a heterogeneous species of cells, possibly not responsive to classic chemotherapeutic strategies. Thus, in addition to being used as a potential biomarker, the possibility of molecular characterisation of the cells might pave the way for therapy specifically targeting such cells, since current treatment options seem to offer limited efficacy with respect to eradicating and controlling this type of disseminated disease. We previously investigated the presence of disseminated tumour cells in BM (DTC) in 316 patients with assumed CRC using immunomagnetic selection (IMS) with the anti-EpCAM antibody MOC31. Disseminated tumour cells were detected in 17% of patients with CRC with increasing frequency through TNM stages 1 -3. In the present work, we present longterm follow-up for this patient cohort, and additionally, we report results obtained by immunocytochemistry (ICC) with anticytokeratin antibodies. # Patients and methods ## Patients Patients undergoing surgery for assumed or verified CRC were included consecutively from five hospitals in the Oslo region between September 1998 and July 2000. The study was approved by the Regional Ethics Committee (Health Region II, Norway, reference no. S-98080), and patient informed consent was obtained in accordance with the Helsinki Declaration. Bone marrow was collected at primary surgery from both anterior iliac crests from 316 patients. Eighty-one patients were excluded from the analysis, leaving a study population of 235 patients (not invasive cancer (n ¼ 25); insufficient material for analysis (n ¼ 2); previous epithelial cancer (n ¼ 7); histology other than adenocarcinoma (n ¼ 5); neoadjuvant chemoradiotherapy (n ¼ 2); incomplete surgical resection (n ¼ 7); or metastases detected at the time of surgery (n ¼ 33)). Follow-up data were obtained from consecutive reports from physicians at participating hospitals. Valid observations of the presence or absence of distant metastases required radiological examination. For patients not attending scheduled controls, data were retrieved from patient records or by contacting the patients' general practitioner. In addition, survival data were obtained from the National Registry of Norway and updated by 1 October 2008. The cause of death was classified as death from CRC, death of other causes or death of unknown cause. For overall survival, median follow-up of patients still alive was 9.3 years (range 8.3 -10.2). Histological evaluation of resected specimens was performed in four pathology laboratories, and to ensure consistent staging and grading, one of the study pathologists (JMN) reevaluated reports and primary sections, simultaneously reassessing the presence or absence of lymphocyte infiltration, vascular and perineural invasion and perinodal growth [fig_ref] Table 1: Baseline clinicopathological characteristics of the study cohort [/fig_ref]. ## Immunomagnetic selection with an anti-epcam antibody Immunomagnetic selection was performed as previously described. Briefly, mononuclear cells (MNCs) were separated from BM by gradient centrifugation using CPT tubes (Becton Dickinson Co., Franklin Lakes, NJ, USA), washed once and resuspended in PBS with 1% HSA (Octapharma AG, Ziegelbrücke, Switzerland). Dynabeads M450 rat antimouse IgG1 or M450 sheep antimouse IgG (Dynal, Oslo, Norway) coated with MOC31 antibody (IQ Products, Groningen, The Netherlands) were added to 2 Â 10 7 MNCs at a ratio of one bead to two MNCs in a total volume of 1 ml in 10 ml, round-bottomed test tubes. For control experiments, uncoated beads were used. Samples were incubated at continuous rotation at 41C for 30 min, and the suspension was then exposed to a strong magnet, and the supernatant was decanted off. Fractions of the remaining cell suspension were examined in a light microscope for the presence of rosetted cells. A sample was classified as positive if a minimum of two cells rosetted at least five beads with the MOC31 antibody, and no rosettes were observed with the control beads. ## Immunocytochemistry with anti-cytokeratin antibodies Direct cytospins were prepared by centrifuging MNC isolated from BM onto glass slides using a Hettich cytocentrifuge (Tutlingen, Germany). The slides were air-dried overnight and stored at À801C. Immunocytochemistry was performed as described previously by incubating four slides (2 Â 10 6 MNC) with anticytokeratin monoclonal antibodies AE1 and AE3 (Sanbio, Uden, The Netherlands) and the same number of slides were incubated with an isotype-specific irrelevant control antibody (MOPC-21; Sigma Chemical Co., St Louis, MO, USA). Slides were evaluated by light microscopy and cytokeratin-positive cells were subjected to strict morphological classification by a pathologist (EB) according to standardised criteria [bib_ref] Standardization of the immunocytochemical detection of cancer cells in BM and blood:..., Borgen [/bib_ref] [bib_ref] A concept for the standardized detection of disseminated tumor cells in bone..., Fehm [/bib_ref]. # Statistical analysis Associations between the presence of DTC and clinicopathological variables were tested using two-tailed Fisher's exact test or linearby-linear association w 2 -test. Univariate survival analysis was performed according to the Kaplan -Meier method, and survival was compared using the log rank test. Multivariate analysis was conducted using the Cox proportional hazards regression model with backward, stepwise elimination of variables. Survival was measured from the date of surgery until death for overall and disease-specific survival, and from the date of surgery until diagnosis of distant metastasis for metastasis-free survival. Data analysis was performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). P-values o0.05 were considered statistically significant. # Results ## Patient characteristics and outcome Clinical and histological parameters of the study population are summarised in [fig_ref] Table 1: Baseline clinicopathological characteristics of the study cohort [/fig_ref]. Mean age at the time of surgery was 72 years (range 35 -98 years). Twenty-nine patients in TNM stage 3 (40%) and two patients in stage 2 (2%) received adjuvant chemotherapy. Tumour perforation at surgery occurred in three rectal cancer patients (two with stage 2 and one stage 3 tumour), and adjuvant radiotherapy was given in these cases. Only 8 of the 69 patients with DTC received adjuvant chemotherapy. Outcome parameters are presented in [fig_ref] Table 2: Detection of disseminated tumour cells and patient outcome parameters [/fig_ref]. Fifty-five patients developed distant metastases during follow-up (after median 15 months, range 1 -59 months), 31 of which had colon cancer and 24 had rectal cancer. A total of 51 CRC-related deaths were registered, and 48 of these patients died of metastatic disease, whereas 3 died of local recurrence. (A discrepancy is apparent between present results and published data, as two IMS negative patients previously incorrectly registered as TNM stage 2 have been corrected to TNM stage 1 (as these corrections represented changes at the decimal level, no erratum was published).) ## Detection of dtc Using the IMS method, EpCAM-positive tumour cells were detected in 41 BM samples (17%) with increasing frequency through TNM stages 1 -3; 10%, 18% and 22%, respectively. A median number of 8 tumour cells (range 2 -120) were detected in positive samples. Cytokeratin-positive cells were detected in 28 samples (12%) with relatively low detection rate (8%) in TNM stage 2, whereas 14% and 17% of samples were positive in stages 1 and 3, respectively. The median number of tumour cells detected by ICC was 1 (range 1 -30), and in 21 of the 28 positive samples only one tumour cell was observed. Of note, only five patients were positive with both methods (one in TNM stage 2 and four in stage 3 patients). The presence of DTC was not associated with any of the clinicopathological variables studied [fig_ref] Table 3: Associations between detection of disseminated tumour cells in bone marrow and clinicopathological... [/fig_ref]. ## Clinicopathological parameters and patient outcome In univariate analysis, TNM stage, lymph node metastases, tumour localisation in the rectum, absence of tumour lymphocyte infiltration and presence of vascular invasion were associated with metastasis development [fig_ref] Table 4: Survival analyses of clinicopathological parameters and disseminated tumour cells in bone marrow [/fig_ref]. The presence of lymph node metastases was the main contributor to the prognostic impact of TNM stage, which was also associated with diseasespecific and overall survival, while pT-stage was not associated with any of the included outcome parameters. The prognostic relevance of tumour localisation was confined to the distinction between colon and rectum. No significant prognostic differences were observed between the presence of left-sided and right-sided tumours (data not shown). Lymphocyte infiltration, vascular invasion and perineural invasion were associated with diseasespecific survival but not with overall survival. ## Detection of dtc and patient outcome The detection of tumour cells in BM was associated with adverse prognosis in this cohort of CRC patients ; [fig_ref] Table 4: Survival analyses of clinicopathological parameters and disseminated tumour cells in bone marrow [/fig_ref]. The presence of EpCAM-positive cells, as detected by IMS, was inversely associated with metastasis-free, disease-specific and overall survival in univariate analyses (P-values 0.049, 0.03 and 0.02, respectively). Immunocytochemistry detection of cytokeratin-positive cells was inversely associated with metastasis-free and disease-specific survival, but not with overall survival (P-values 0.03, 0.002 and 0.06, respectively). In multivariate Cox regression analyses, the impact of detecting EpCAM-positive cells in BM remained evident for metastasis-free survival [fig_ref] Table 4: Survival analyses of clinicopathological parameters and disseminated tumour cells in bone marrow [/fig_ref] , as well as for disease-specific (P ¼ 0.002; HR ¼ 3.0; CI ¼ 1.5 -5.8) and overall survival (P ¼ 0.006; HR ¼ 1.9; CI ¼ 1.2 -3.0). For cytokeratinpositive cells, an association was preserved in multivariate analysis only for disease-specific survival (P ¼ 0.01; HR ¼ 2.5; CI ¼ 1.2-5.0). Additional analyses were then performed to assess whether the influence of DTC had particular relevance in classic prognostic subgroups defined by the TNM staging system. In univariate analyses, the prognostic significance of IMS-detected EpCAM-positive cells was evident in TNM stage 3, whereas significant impact of ICC-detected cytokeratin-positive cells was confined to TNM stage 2 . # Discussion The main finding of this prospective study was that the presence of DTC was a prognostic biomarker in this cohort of curatively resected patients with CRC. Surprisingly, the use of IMS with an anti-EpCAM antibody and ICC with anti-cytokeratin antibodies for detection of tumour cells resulted in minimal detection overlap, although results obtained with each method were associated with outcome in distinct prognostic subgroups of CRC patients. The detection rate of EpCAM-and cytokeratin-positive samples in this study (in 17% and 12% of cases, respectively) was somewhat lower than previously reported. A recent meta-analysis identified only six studies that investigated the relevance of DTC in relation to robust outcome parameters, such as metastasis-free and overall survival [bib_ref] Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in..., Rahbari [/bib_ref]. Of these studies, five utilised ICC and one RT -PCR detection strategies in cohorts comprising 61 -167 patients, and DTC were detected in 24 -61% of cases. Although suggesting a clinical relevance in CRC, the predictive value of DTC could not be unambiguously determined based on the results reported in these studies. The present work comprises one of the largest CRC patient cohorts examined for the presence of DTC, employing in parallel two distinct detection methods. Although applying the combined results of two detection methods, only 21 of the 55 patients that developed overt metastases could be identified. Conversely, 46 of the 180 patients that did not develop metastatic disease had DTC (combining results from the two methods), giving test specificity and sensitivity for prediction of metastasis development of 74% and 38%, respectively. This infers that in more than half of the metastatic patients, tumour cells were either not present in BM at diagnosis, or EpCAM and cytokeratin expression levels were below the detection limit of the respective method. The finding that detected cells in many cases did not give rise to secondary tumour formation is in turn not surprising, given the known inefficiency of the metastatic process [bib_ref] Metastasis: dissemination and growth of cancer cells in metastatic sites, Chambers [/bib_ref]. The high detection rate of DTC remains in intriguing contrast to the relatively low incidence of overt bone metastases in CRC [bib_ref] Metastasis: dissemination and growth of cancer cells in metastatic sites, Chambers [/bib_ref] (in the present study bone metastases were reported in 3 of 55 patients with metastatic disease), suggesting that BM may mirror dissemination of tumour cells to typical metastatic sites (liver and lungs) or alternatively, represent a reservoir of tumour cells able to initiate growth in secondary organs. The kinetics of overt metastasis development (here observed to occur at median 15 months, range 1-59 months after primary surgery) further suggests that DTC may be representative of dormant tumour cells with the ability to escape dormancy control to self-renew and differentiate upon receiving an appropriate stimulus . While analysis of cytokeratin expression was chosen primarily for being a hallmark of epithelial cellular origin, EpCAM expression Abbreviation: CI ¼ confidence interval. a Stepwise Cox regression analysis is shown for metastasis-free survival. b Perinodal growth was assessed in node-positive patients only. DTC as a prognostic biomarker in CRC K Flatmark et al might be of additional biological interest. Complementing its functions as a homeotypic cell adhesion molecule in epithelialderived cells, EpCAM has been identified as a mitogenic signal transducer and a stem cell marker [bib_ref] Nuclear signalling by tumourassociated antigen EpCAM, Maetzel [/bib_ref] [bib_ref] The emerging role of EpCAM in cancer and stem cell signaling, Munz [/bib_ref]. Thus, detection of EpCAM-positive tumour cells could signify the presence of cells with stem-like properties in BM, potentially contributing to the observed association with metastasis development and patient survival. Interestingly, administration of adjuvant chemotherapy did not influence metastasis development and survival in this patient cohort (data not shown). Although the study design could explain lack of power to detect differences that might still be present, the data may also reflect that the chemotherapy regimen used in the adjuvant setting at that time (5-fluorouracil and calcium folinate) may have been inefficacious. It remains of vital importance to identify and characterise cells responsible for metastatic relapse at the molecular level, with the potential reward of identifying strategies to target therapy towards the prolongation of dormancy or to attain cell death [bib_ref] Maintenance therapy to suppress micrometastasis: the new challenge for adjuvant cancer treatment, Epstein [/bib_ref]. The low degree of positive overlap observed between the two detection methods are in accordance with a recent study of 391 early breast cancer patients, in which positive diagnostic overlap of only 3.2% was observed when ICC was performed using two different pan-anti-cytokeratin antibodies, and distinct clinical implications were associated with each antibody [bib_ref] Detection and clinical relevance of early disseminated breast cancer cells depend on..., Effenberger [/bib_ref]. In our study, the presence of EpCAM-and cytokeratin-positive cells were prognostic predictors in separate subgroups of CRC patients, in TNM stages 3 and 2, respectively. It is difficult to provide a biological explanation for this finding, and a likely cause could be the low number of cases remaining for calculations in the subgroup analyses. Substantiating the findings of these clinical trials are studies characterising single or small numbers of DTC reporting considerable heterogeneity with respect to genotypic and phenotypic properties of cells from the same individual, reflecting the heterogeneity of the primary tumour [bib_ref] Genomic profiling of viable and proliferative micrometastatic cells from earlystage breast cancer..., Gangnus [/bib_ref]. Thus, a possible explanation for the lack of overlap between methods could be the presence of subpopulations of tumour cells in BM with distinct molecular characteristics, and to our knowledge, this is the first report to demonstrate clinical impact of such findings in CRC. Even counting for publication bias, the number of reports supporting a clinical relevance for DTC in solid tumours is remarkable, comprising most solid tumour forms [bib_ref] Cancer micrometastases, Pantel [/bib_ref]. Thus, it seems probable that tumour cells are detected, although for most analytical methods, actual proof cannot be provided for each detected cell. The present results add to the evidence suggesting a role for DTC detection as a biomarker in CRC. However, although biologically interesting, the variable results obtained with different detection methods raise more questions than are answered, rendering available technology immature to the extent that it cannot be implemented in routine clinical practice at present. Emerging results from characterisation studies as well as from clinical trials like our own suggest that a detection method suitable for clinical use should be able to account for the apparent heterogeneity of DTC and its relevance in the clinical setting. To address these questions, DTC detection using a range of detection methods should be included in future clinical trials, preferably in the adjuvant setting. This would allow assessment of predictive robustness on a larger scale, determination of potential associations with treatment response as well as facilitate studies of relevant subpopulations of tumour cells with the possibility of correlating results to clinical parameters. [fig] Figure 1, Figure 2: Kaplan -Meier survival plots demonstrating the association between the presence of disseminated tumour cells in bone marrow and survival end points. Immunomagnetic selection (IMS) with an anti-EpCAM antibody and (A) metastasis-free (B) overall survival. Immunocytochemistry (ICC) with anti-cytokeratin antibody and (C) metastasis-free and (D) overall survival. Kaplan -Meier survival plots demonstrating the association between the presence of disseminated tumour cells in bone marrow and metastasisfree survival in stage subgroups at diagnosis. Immunomagnetic selection (IMS) in TNM stages 1 (A), 2 (B) and 3 (C). Immunocytochemistry (ICC) in TNM stages 1 (D), 2 (E) and 3 (F). [/fig] [table] Table 2: Detection of disseminated tumour cells and patient outcome parameters [/table] [table] Table 3: Associations between detection of disseminated tumour cells in bone marrow and clinicopathological parametersAbbreviations: ICC ¼ immunocytochemistry; IMS ¼ immunomagnetic selection; NA ¼ not applicable. a P-values are from Fisher's exact test or linear-by-linear association w 2 -test. b Perinodal growth was assessed in node-positive patients only. [/table] [table] Table 1: Baseline clinicopathological characteristics of the study cohort [/table] [table] Table 4: Survival analyses of clinicopathological parameters and disseminated tumour cells in bone marrow [/table]

Metastatic HPV‐related oropharyngeal carcinoma cured with chemoradiotherapy: importance of pretherapy biomolecular assessment Pretherapy assessment has a crucial role in the management of advanced oropharyngeal carcinoma. The case report represents an example of how translational research may help to optimize the therapeutic options and to choose a well-shaped therapy adapted to the tumor and the patient.KeywordsCyclin D1, human papillomavirus, oropharyngeal carcinoma, P16, p53. # Introduction Oropharyngeal carcinomas (OCs) account for 20% of squamous cell carcinomas of the head and neck (SCCHN), and their incidence is steadily increasing [bib_ref] The growing epidemic of HPV-positive oropharyngeal carcinoma: a clinical review for primary..., Moore [/bib_ref]. OCs often are diagnosed as locally advanced (LA) disease, namely staged as T1N1 until T4N3 according to American Joint Committee against Cancer (AJCC) staging system. Locally advanced oropharyngeal carcinomas (LAOCs) are currently managed with concomitant cisplatin and radiation therapy, according to the National Comprehensive Cancer Network (NCCN) guidelines, and upfront surgery is seldom necessary, being recommended only for very advanced T4 lesions [bib_ref] Induction chemotherapy with concurrent chemoradiotherapy versus concurrent chemoradiotherapy for locally advanced squamous..., Zhang [/bib_ref]. Conservative strategy represents the preferred treatment option for LAOCs, being most of them characterized by good response to both chemo-and radiotherapy. Nevertheless, a number of patients treated with chemoradiotherapy often experience recurrent disease and show poor prognosis [bib_ref] Treatment outcomes for T4 oropharyngeal squamous cell carcinoma, Zenga [/bib_ref]. Progressive disease or early recurrence after chemoradiation may characterize some chemo-and radio-resistant forms, which show poor prognosis and scarce response to conservative treatments [bib_ref] Radioresistant head and neck squamous cell carcinoma cells: intracellular signaling, putative biomarkers..., Skvortsov [/bib_ref] [bib_ref] Chemoradiation for advanced oral and (pharyngo) laryngeal carcinoma: single institution outcome analysis, Dequanter [/bib_ref]. Recently, an increase in diagnosis of some OCs has been documented, especially those arising from tonsil and base of tongue. This phenomenon has been associated with the increased incidence of human papillomavirus (HPV)-positive tumors [bib_ref] Detection of HPV-associated oropharyngeal tumours in a 16-year cohort: more than meets..., Melchers [/bib_ref]. HPV-positive OCs generally show good prognosis and, in some reports, also a better response to chemo-and radiotherapy, when compared with the HPV-negative counterparts [bib_ref] Human papillomavirus and survival of patients with oropharyngeal cancer, Ang [/bib_ref] [bib_ref] De-escalation treatment protocols for human papillomavirus-associated oropharyngeal squamous cell carcinoma, Masterson [/bib_ref]. This last feature has paved the way to a number of clinical trials aimed to de-intensify standard treatments, substituting, for example, cisplatin with cetuximab given 56 concomitantly to radiotherapy, or removing definitively surgery from the therapeutic armamentarium. Nevertheless, some HPV-positive OCs show a grim prognosis and a poor response to chemoradiotherapy, especially if diagnosed in strong smokers or drinkers and in the 6th or 7th decade of age [bib_ref] Human papillomavirus and survival of patients with oropharyngeal cancer, Ang [/bib_ref] [bib_ref] Discrimination of 'driver' and 'passenger' HPV in tonsillar carcinomas by the polymerase..., Evans [/bib_ref]. One interpretation is that not all the HPV-positive tumors are also HPV-related neoplasms. HPV-driven cancerogenesis leads to the onset of tumors characterized by special features, such as cell cycle disruption, high proliferating index, and, consequently, high sensitivity to both chemo-and radiotherapy. Moreover, HPV-related tumors often are diagnosed in the young adult, nonsmokers, or slightly smokers, with no anamnesis of alcohol consumption and with history of several sexual partners [bib_ref] P16 (INK4a) correlates to human papillomavirus presence, response to radiotherapy and clinical..., Mellin Dahlstrand [/bib_ref] [bib_ref] HPV-related oropharyngeal cancers: from pathogenesis to new therapeutic approaches, Tornesello [/bib_ref]. HPV-related OCs show p16 and p21 overexpression, TP53 not mutated, CCND1 wild-type status, and low epidermal growth factor receptor (EGFR) expression at immunostaining. The tobaccoand alcohol-related OCs, namely the HPV-negative counterpart, often show the opposite features [bib_ref] Radioresistance in head and neck squamous cell carcinoma: biological bases and therapeutic..., Perri [/bib_ref] [bib_ref] Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity..., Raju [/bib_ref] [bib_ref] TP53 disruptive mutations lead to head and neck cancer treatment failure through..., Skinner [/bib_ref] [bib_ref] Cyclin D1 overexpression is associated with poor prognosis in oropharyngeal cancer, Lin [/bib_ref]. In the future, it is likely that making a distinction between HPV-and non-HPV-related OCs will be of utmost importance, being the first very chemo-and radiosensitive, and thus more suitable for conservative treatments, even if diagnosed at advanced stage. ## Case presentation About 32 months ago, a 39-year-old Caucasian male patient came to our attention, suffering from dysphagia and odynophagia which were persistent after several lines of antibiotics therapy. He referred us to be a slight smoker (<10 cigarettes in a month) and did not report alcohol consumption. At a clinical examination, a wide lesion arising from the right tonsil was detected, and at the neck examination, an omolateral laterocervical mass was found interesting the II level according to Robbins classification. A fibroscopy confirmed the presence of a wide exophytic mass arising from the right tonsil and infiltrating the omolateral tonsillar pillar. A biopsy of the lesion was performed during the fibroscopy. Histopathologic examination of the biopsy sample confirmed the clinical suspicion, identifying a poorly differentiated squamous cell carcinoma of the right tonsil. The neoplasm was staged by performing a computed tomography of the head, neck, thorax, and abdomen, which better defined the primary tumor extension. The mass measured 5 centimeters in the biggest diameter and infiltrated the base of tongue and the floor of mouth. A single 2,5 centimeters lymph node metastasis was detected at the station II of the laterocervical chain. Moreover, multiple bilateral lung lesions were detected, the biggest of them being 1,5 centimeters length. A following Positron emission tomography (PET/TC) confirmed the malignant nature of the oropharyngeal lesion, which appeared as an area showing high uptake (SUV 12.6); both the lymph node (SUV: 10) and lung lesions (SUV: 6) also strongly pick up the FDG (fluoro-deoxy-glucose) [fig_ref] Figure 1: June 2013 [/fig_ref]. Stage of disease was T4N1M1 (IVc according to AJCC). ## Additional determinations Histopathologic diagnosis was a poorly differentiated squamous cell carcinoma. The HPV of the neoplasm was studied by in situ hybridization with positive result. A number of biological features were studied by immunohistochemistry, such as p16, p21, EGFR, TP53, Akt, and CCND1 status. Immunohistochemistry was employed to detect EGFR, P16, P21, Cyclin D1 (which is the product of CCND1 gene), and phospho-Akt expression. Polymerase chain reaction (PCR) was used to identify mutations in the TP53 gene. As a result, p16 and p21 were overexpressed, while EGFR, phospho-Akt, and Cyclin D1 were not. No TP53 mutations were detected. Immunohistochemistry was performed on representative 4 -lm sections cut from formalin-fixed, paraffinembedded tissue blocks, using a monoclonal antibody to p16 (MTM Laboratories; monoclonal; 1:1 dilution) on a Ventana Benchmark LT automated immunostainer (Ventana Medical Systems, Tucson AZ) according to standard protocols. P16-positive samples have been considered all that displaying a tissue expression of at least 51%. Primary antibody incubation was carried out overnight at room temperature using a mouse monoclonal antibody for Cyclin D1 (Clone DCS-6, DAKO Corp. A/S, Glostrup, Denmark) at 1:50 dilution. We graded the distribution of positive cells in tumors that stained for Cyclin D1 as follows: grade 1, focal staining in <10% of tumor cells; grade 2, fairly widespread staining in 10-50% of tumor cells; and grade 3, diffuse staining in more than 50% of tumor cells. We considered as "overexpressed" values above 50%. Epidermal growth factor receptor protein expression was assessed by IHC with the Dako EGFR PharmDx kit (DakoCytomation, Berkeley, CA). Samples were classified as EGFR IHC overexpressed if ≥50% of the tumor cells demonstrated membranous staining of any intensity. Detection of TP53 mutations was carried out using the IARC (International Agency for Research on Cancer) database, reporting mutations in several loci included between exons 2 and 11. PCR was employed, and primers located at least 50 bp away from the ends of each exon were designed to amplify exons 1-11 of the TP53 gene. We have used Phospho-Akt (Ser473) (736E11) Rabbit mAb to measure PI3K/Akt/mTor pathway activation in paraffin-embedded tissue samples. The intensity is designated as 0 when no tumor cells stained, 1+ when 10-20% of cells stained (weak), 2+ when 20-50% of cells stained (moderate), and 3+ when >50% of cells stained (strong). Santa Cruz Biotechnology p21 antibody (Santa, sc-6246) was used. The intensity of the staining was scored as follows: low when 1-10% of cells showed positive staining, moderate when 11-50% of them showed positive staining, and severe when more than 50% showed positive staining. ## Treatment Given the disease extension (IVc sec AJCC), a polychemotherapy regimen, based on cisplatin-cetuximab-5-fluorouracil, was recommended by American and European guidelines. Nevertheless, on the basis of HPV positivity, with wild-type Cyclin D1, p16, and p21 overexpressed, TP53 wild type and EGFR slightly expressed, we could expect a good response to chemo-and radiotherapy. Thus, we chose to employ induction chemotherapy conditionally followed by chemoradiotherapy, in case of complete response or complete disappearance of lung metastases. We chose to use a strongly active scheme, and 3 months after the first diagnosis, three cycles of cisplatin-docetaxel, as induction chemotherapy, were administered. Both cisplatin and docetaxel were employed at a dose of 80 mg/m2 on day one, and they were repeated every 3 weeks. Acute toxicity was mild being grade 2 neutropenia after the third cycle, and grade 3 alopecia and grade 1 nausea after 2 weeks from the start of chemotherapy. ## First restaging Two months after the first chemotherapy cycle, a restaging CT scan was performed, and as a result, complete disappearance of lung lesions as well as the oropharyngeal mass was observed. The lymph node lesion appeared strongly reduced when compared with previous CT scan [fig_ref] Figure 2: September 2013 [/fig_ref]. A PET/TC was also performed, and it confirmed the CT scan findings, and as a result, only the oropharyngeal lesion persisted, but its uptake was strongly reduced (SUV 2.2 vs. 12.6). ## Treatment On the basis of the optimal response obtained after induction chemotherapy, as well as the complete disappearance of distant lesions, we chose to continue the therapeutic program and to administer concurrent chemoradiation. External beam radiotherapy was administered using 6 MV of energy photons of the linear accelerator (LINAC). A 3D conformal technique was employed, and a total dose of 70 Gy was reached using a conventional fractionating regimen (2.00 Gy per fraction). The target to be irradiated was considered to be the primary tumor plus bilateral laterocervical lymph nodes starting from level Ib to level IV. This last level was considered as the CTV (clinical target volume). A margin of 1 centimeter was added to CTV with the aim of avoiding positioning errors and involuntary patient movements, and as a result, a larger volume was obtained, named PTV (planning target volume). More in particular, we delineated a PTV1, which comprised the gross tumor volume (GTV) in the oropharynx plus the bilateral lymph node stations from Ib to IV, and we administered a maximum dose of 60 Gy on this field. Then, we identified a PTV2, which comprised only the oropharyngeal mass and the visible lymph nodal lesion, in the right laterocervical region. We reached a total dose of 66 Gy on PTV2. Finally, we designed a third and shorter treatment volume, namely PTV3, which included only the gross oropharyngeal tumor volume, and we reached the total 70 Gy dose on it. For CTV delineation, we used prechemotherapy tumor extension. Concomitant chemotherapy was performed employing cisplatin at dose of 80 mg for square meter of body surface given every 3 weeks. The aforementioned chemoradiation protocol was administered for a duration of 7 weeks, and it was started 25 days after chemotherapy completion. Acute toxicity was moderate, being grade 3 dysphagia the worse side effect encountered. It appeared after 5 weeks from the start of chemoradiation and lasted throughout the treatment, requiring also the feeding tube apposition. Grade 2 nausea and skin erythema started during the second week of treatment and lasted about 1 month. No further side effects were seen after the completion of the chemoradiation. ## Second restaging One month later, a CT scan was performed, and as a result, only a modest asymmetry of the oropharyngeal cavity was detected, without visible lesions or contrast enhancement. Neither laterocervical norlung masses were seen. Two months later, a PET/TC did not detect any pathologic uptake in the examined body parts explored. In addition, we performed a complete clinical examination and a fibroscopy that did not show suspicious lesions or features. We judged the patient to be in complete response (CR) [fig_ref] Figure 3: February 2014 [/fig_ref] , and we addressed him to a follow-up program. ## Follow-up A first follow-up visit was made 3 months later and consisted in a CT scan and a complete clinical examination. Fibroscopy did not detect suspicious lesions. A following CT scan was performed 4 months later and did not highlight significant features. A following CT scan was performed 6 months later and did not detect pathological features. The last CT scan was performed 6 months later and was considered as negative for disease recurrence. Actually, the patient is alive, and at a last follow-up visit, on December 2016, he was recurrence-free. # Discussion Epidemiology of SCCHNs has profoundly changed in the last decade, mainly due to the increasing frequency of virus-related malignancies [bib_ref] De-escalation treatment protocols for human papillomavirus-associated oropharyngeal squamous cell carcinoma, Masterson [/bib_ref]. HPV infects the upper aerodigestive tract and the proximal airways, showing high tropism for some areas, among oropharynx. OCs, but also other HPV-related SCCHNs, are characterized by peculiar features that distinguish them significantly from those non-HPV-related. In particular, the cancerogenesis process in head and neck cancer, among oropharyngeal cancer, is mainly due to the mutagens present in tobacco and alcohol, which may alter DNA in specific loci leading to neoplastic progression [bib_ref] P21 Cip1/ WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and..., Hafkamp [/bib_ref]. HPV-related cancerogenesis is very different, and HPV-related malignancies are characterized by good prognosis and good response to both chemo-and radiotherapy in clinical trials [bib_ref] CCND1 as a predictive biomarker of neoadjuvant chemotherapy in patients with locally..., Feng [/bib_ref] [bib_ref] Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset..., Posner [/bib_ref]. Given their chemoand radiosensitivity, HPV-related head and neck carcinomas could be treated preferably with conservative approaches such as sequential or concurrent chemoradiotherapy. Nevertheless, it is crucial to distinguish between HPV-positive and HPV-related tumors. In fact, some neoplasms which show positivity for HPV are characterized in any case by poor prognosis and scarce response to both chemo-and radiotherapy, because they arise in heavy smokers and/or drinkers. In this last case, the impact of HPV on cancerogenesis is only marginal. On the other hand, when HPV is the main promoter of cancerogenesis, tumor cells show typical features, such as high proliferating index, a low number of DNA changes, and high sensitivity to either chemical mutagens or ionizing radiations. The immunohistochemical panel, which we have chosen to adopt, has led us to identify a tumor strongly related to HPV effect. This type of cancerogenesis is strongly HPV-driven, and the neoplasm which has followed is markedly chemo-and radiosensitive. For this reason, we have treated the patient with the aim of curing him even if he has a metastatic disease, choosing to avoid palliative cetuximab-based polychemotherapy, in favor of sequential chemoradiotherapy. As a result, patient obtained a complete response yet after induction chemotherapy and we have consolidated that response performing a concurrent chemoradiotherapy protocol. Ongoing clinical trials are evaluating the possibility to select the patients with the aim to assess their chemoand radiosensitivity. Some reports support the use of a conservative treatment in HPV-related malignancies, given their sensitivity to chemotherapy and radiotherapy. # Conclusion Metastatic head and neck carcinomas are often associated with poor prognosis, and the standard therapy is the palliative chemotherapy, which is associated with a median overall survival of 12 months in clinical trials. The abovementioned case report might be an example of the best application of translational research in oncology, namely the pretherapy study of the specific tumor-associated "signature" and the employment of a well-shaped therapy conformed to this "signature." Lately, a well-defined entity, namely HPV-associated head and neck carcinomas, has been identified, and patients affected by HPV-related tumors show a good chemo-and radiosensitivity. In the future, we can try to cure these patients albeit metastatic, using chemo-and radiotherapy in the right sequence. # Consent statement Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review with the Editor-in-Chief of this journal. # Informed consent statement The patient provided us a written informed consent. [fig] Figure 1: June 2013; first staging with computed tomography. 58 ª 2017 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. [/fig] [fig] Figure 2: September 2013; first restaging after induction chemotherapy. ª 2017 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. [/fig] [fig] Figure 3: February 2014; second restaging after chemoradiation (CT scan). [/fig]

A Case Report of Lifesaving Intravenous Bolus Epinephrine Administration in a Case of Severe Refractory Anaphylactic Shock # Introduction Anaphylaxis is an acute hypersensitivity reaction to a stimulus that can be life-threatening if early measures are not taken promptly [bib_ref] The epidemiology of anaphylaxis, Yu [/bib_ref]. The prevalence of anaphylaxis is 0.05-2% and 3% in the United States of America and Europe, respectively [bib_ref] The epidemiology of anaphylaxis, Yu [/bib_ref]. It can be triggered by various factors such as food, medications, insect stings, and many other agents [bib_ref] The epidemiology of anaphylaxis, Yu [/bib_ref]. Patients can exhibit various systematic signs and symptoms that can range from mild reactions with skin manifestations to severe life-threatening reactions such as respiratory compromise and hypotension [bib_ref] The epidemiology of anaphylaxis, Yu [/bib_ref]. Early recognition of anaphylaxis clinical presentation and early medical intervention is vital in reducing the chances of developing anaphylactic shock. Reversing anaphylaxis can be achieved by removing the causative agent if possible, assessing the airway, breathing, circulation, and injecting intramuscular (IM) epinephrine repeatedly as needed [bib_ref] World Allergy Organization Anaphylaxis Guidance 2020, Cardona [/bib_ref]. In this report, we would like to emphasize the importance of rapid and early stabilization of patients with anaphylaxis and highlight our approach to overcoming and successfully managing a case of severe anaphylactic shock refractory to standard first-line therapy. ## Case presentation A 31-year-old Pakistani male with a known case of hypertension for 2 years presented to the emergency department complaining of nausea, vomiting, right flank pain, and headache. The patient reports noncompliance with his therapeutic regimen as he is uninsured and cannot afford his medications. The right flank pain started one day ago and was associated with nausea and three episodes of vomiting. The vomitus was of normal food content, non-bloody, non-projectile, and non-bilious. The patient has a onemonth history of recurrent moderate, intermittent right flank pain radiating to the right groin worsened with urination and is not associated with fever or rigors. Also, the headache started in the last 2 months, worsening in the last week and it was described as a generalized headache, moderate in severity, and not associated with changes in vision or photophobia. The patient is not following up with his general practitioner and is not currently taking any medications, has not had any previous surgeries, has no known allergies, and does not report any insect bites. He works an office job and does not smoke or drink alcohol. Family history is positive for hypertension on the paternal side. On examination, the patient looked well, alert, oriented, and not in distress. The patient had mild tenderness on head palpation and over the right flank. His vital signs were as follows: temperature 37°C, blood pressure (BP) 212/134 mmHg, heart rate 78 beats/minute, respiratory rate 12 breaths/minute, and oxygen saturation 100% on room air. The patient's weight and height were 75 kg and 1.7 m, respectively with a calculated body mass index of 25.95 kg/m 2 . The patient was triaged to level 4 care and was started on a stat dose of captopril 12.5 mg tablet. His BP lowered to 191/116 mmHg over a period of 1 hour. A stat dose of amlodipine 5 mg tablet was given to help lower his BP further along with a stat dose of diclofenac 75 mg (1 mg/kg) intramuscular (IM) for his flank pain. Two minutes later, the patient started getting agitated and the nurses noticed that the patient started developing swelling of his mucosal membranes, no urticaria, or rashes were visualized and his BP suddenly dropped and became unrecordable. The patient was immediately given two standard adult doses of IM epinephrine injections for anaphylaxis into the right deltoid of 500 micrograms each with a 5minute interval (0.5 mL of 1 mg/mL, 1:1000 epinephrine), two large-bore cannulas started and 1 L of normal saline (NS) was administered, the patient was placed on high-flow oxygen, stat IV hydrocortisone 300 mg (4 mg/kg), stat IV promethazine 25 mg (0.33 mg/kg), and albuterol nebulization of 1 mL over 5 minutes was given three times back-to-back. However, his BP did not pick up. The patient was immediately shifted to level 2 care where he was connected to a cardiac monitor and a continuous pulse oximeter and given a third IM epinephrine injection of 500 mcg. An IV epinephrine drip was started at a rate of 4 drops/min (1 mg of epinephrine 1:1000 in 1 L NS i.e., 4 mcg/min). The patient's BP reached 60/40 mmHg. However, it was not maintained and was progressively dropping down. A decision by the team leader was to administer an IV epinephrine bolus of 300 mcg (3 mL stat of (1 mL of 1/10,000 epinephrine + 9 mL of NS) i.e., 4 mcg/kg). The patient's BP raised to 126/75. After 10 minutes, the BP dropped again to 88/59 mmHg, hence giving another IV epinephrine bolus of 200 mcg (2 mL stat of (1 mL of 1/10,000 epinephrine + 9 mL of NS) i.e., 2.6 mcg/kg). His BP picked up to 140/90 mmHg. The patient started shivering and his lips and nose swelling started to resolve. Ear-nose and throat and intensive care unit on-call doctors were consulted immediately following the recognition of this anaphylaxis. No airway obstruction was visualized and intubation was not necessary. A summary of the timeline of events that happened during this case is summarized in [fig_ref] TABLE 1: Timeline demonstrating the interventions and corresponding vitals of the patient following administering... [/fig_ref] and the laboratory investigations following the anaphylactic episode are summarized in [fig_ref] TABLE 2: Laboratory investigations following the anaphylactic episode [/fig_ref]. Later after stabilization of the patient's condition, he was admitted for observation where he achieved full recovery. The patient was advised to increase fluid intake and was discharged a day later on valsartan-hydrochlorothiazide 160-12.5 mg tablet OD PO, nifedipine 30 mg tablet OD PO, metoclopramide hydrochloride 10 mg tablet TDS for 5 days, and paracetamol 500 mg tablet PRN. # Discussion In this case report, we discuss the successful management of a patient in severe anaphylactic shock with signs of cardiovascular compromise that has not responded to first-line management and where rescue measures were required to save the patient and prevent imminent collapse. The patient received three medications that could act as culprits to his anaphylactic shock namely: captopril, amlodipine, and diclofenac. Angiotensin-converting enzyme inhibitors (captopril) and calcium channel blockers (amlodipine) are considered the first-line management of hypertension in certain patient demographics [bib_ref] The combination of an ACE inhibitor and a calcium channel blocker is..., Widimský [/bib_ref]. Diclofenac is a widely used non-steroidal anti-inflammatory drug (NSAID) in the management of pain, especially in renal colic as it has been heavily researched and has the strongest evidence of effectiveness [bib_ref] Systematic review of the relative efficacy of non-steroidal anti-inflammatory drugs and opioids..., Holdgate [/bib_ref]. These medications are usually safe and effective. NSAIDs have been reported to induce anaphylactic reactions, while angiotensin-converting enzyme inhibitor-induced angioedema is also well documented [bib_ref] Pharmacotherapy for angiotensin-converting enzyme inhibitor-induced angioedema: a systematic review, Lawlor [/bib_ref]. Anaphylactic reactions to amlodipine are exceedingly rare with only a few isolated events reported [bib_ref] Amlodipine-induced angioedema: an unusual complication of a common medication, Kuruvilla [/bib_ref]. Due to the temporal relationship between drug administration and the onset of symptoms, the most likely offender, in this case, is diclofenac as symptoms started to develop almost immediately following its administration. The European Academy of Allergy and Clinical Immunology (EAACI) recently updated the anaphylaxis guidelines 2021 following conducting a comprehensive systemic review of high-quality research. The agreed-upon initial first-line management of anaphylaxis is to assess the airway, breathing, circulation, disability, and exposure (ABCDE) of the patient, remove potential triggers and administer epinephrine. This is then followed by continuous monitoring, escalating to emergency teams with expertise in critical care, and providing supportive measures as needed including administering oxygen, IV fluids, nebulized epinephrine, beta-2 agonists, corticosteroids, and antihistamines. ## Role of epinephrine in anaphylaxis Early administration of epinephrine in anaphylaxis has been shown to reduce both rates of biphasic reactions and overall fatalities. Prompt intramuscular (IM) administration of epinephrine in the midthigh is recommended. In comparison to IM administration of epinephrine in the mid-thigh, it is found that administration of epinephrine in the deltoid or through the use of a metered-dose inhaler or subcutaneously all resulted in lower plasma levels of epinephrine. IV administration of epinephrine resulted in a quicker rise in plasma levels of epinephrine but is associated with an increased risk of epinephrine overdosing and cardiovascular side effects in comparison to IM administration [bib_ref] Epinephrine in anaphylaxis: higher risk of cardiovascular complications and overdose after administration..., Campbell [/bib_ref]. Multiple IM epinephrine injections might be required depending on the patient's response. In some special circumstances including refractory respiratory distress and hypotension, where anaphylaxis is refractory to IM epinephrine, IV epinephrine should be used. Two routes to administer IV epinephrine are through an IV drip or an IV bolus. IV drips are preferred as they are controlled and can be titrated according to the patient's response [bib_ref] Epinephrine in anaphylaxis: higher risk of cardiovascular complications and overdose after administration..., Campbell [/bib_ref]. Bolus epinephrine is usually reserved for refractory cases to all the above measures [bib_ref] Epinephrine in anaphylaxis: higher risk of cardiovascular complications and overdose after administration..., Campbell [/bib_ref]. Furthermore, the use of IV epinephrine should be restricted to healthcare professionals who are trained to use it and to a monitored clinical setting [bib_ref] Epinephrine in anaphylaxis: higher risk of cardiovascular complications and overdose after administration..., Campbell [/bib_ref] [bib_ref] Insect sting anaphylaxis; prospective evaluation of treatment with intravenous adrenaline and volume..., Brown [/bib_ref]. In this case, the patient received three IM epinephrine injections into the right deltoid (the thigh was not accessible due to extremely tight clothing and high BMI) and started on an IV drip without significant improvement. All supportive measures were also administered as a shotgun approach. Despite these interventions, the patient was still in severe grade 4 systemic anaphylactic reaction according to the Mueller grading system. The decision was made by the emergency on-call consultant to administer an IV bolus of epinephrine to prevent imminent cardiovascular collapse. This was done under close monitoring of the patient in triage level 2 and in collaboration with the intensive care unit. The patient received a total of 500 mcg, 300 mcg (3 mL stat of (1 mL of 1/10,000 epinephrine + 9 mL of NS) i.e., 4 mcg/kg) followed by a second 200 mcg (2 mL stat of (1 mL of 1/10,000 epinephrine + 9 mL of NS) i.e., 2.6 mcg/kg) administered 5 minutes apart which helped to normalize the BP. When looking into different routes of epinephrine administration and comparing the maximum plasma concentration (Cmax) and time required to achieve Cmax (Tmax), it has been shown that IM epinephrine administration achieved the highest Cmax when compared to IV, subcutaneous, and inhaled epinephrine administration [bib_ref] Epinephrine absorption after different routes of administration in an animal model, Gu [/bib_ref]. However, when comparing the Tmax required to achieve the Cmax, Cmax was achieved immediately through IV administration while Tmax was on average 32.5, 111.7 , and 45.8 minutes for IM, subcutaneous , and inhaled administration respectively [bib_ref] Epinephrine absorption after different routes of administration in an animal model, Gu [/bib_ref]. Due to the patient being severely hypotensive and at risk of collapse, the delivery of epinephrine from the IM route to the target organs (heart and vascular system) is delayed. Bolus epinephrine provides a means of direct delivery to these organs hence providing the desired effect from the drug in a time-critical fashion. The crucial role epinephrine plays in the management of anaphylaxis is owing to its pharmacological actions. Epinephrine is an endogenous catecholamine which increasingly recognized as an important metabolic hormone released in response to stress [bib_ref] Adrenaline: insights into its metabolic roles in hypoglycaemia and diabetes, Verberne [/bib_ref]. It helps mobilize energy stores and induce a number of changes in the body collectively known as the fight-or-flight response [bib_ref] Adrenaline: insights into its metabolic roles in hypoglycaemia and diabetes, Verberne [/bib_ref] [bib_ref] World allergy organization guidelines for the assessment and management of anaphylaxis, Simons [/bib_ref]. Through its action on alpha-1 adrenergic receptors, epinephrine administration results in increased peripheral vascular resistance, BP, and coronary artery perfusion while lessening vessel vasodilation and increased vascular permeability that occurs during anaphylaxis, which can lead to loss of intravascular fluid volume and hypotension [bib_ref] World allergy organization guidelines for the assessment and management of anaphylaxis, Simons [/bib_ref]. Positive inotropic and chronotropic activity is mediated through its action on beta-1 adrenergic receptors [bib_ref] World allergy organization guidelines for the assessment and management of anaphylaxis, Simons [/bib_ref]. It also increases bronchial smooth muscle relaxation and helps alleviate bronchospasm, wheezing, and dyspnea that may occur during anaphylaxis through its action on beta-2 adrenergic receptors [bib_ref] World allergy organization guidelines for the assessment and management of anaphylaxis, Simons [/bib_ref]. Epinephrine also possesses vasodilator effects in the skeletal muscle which explains why IM administration is preferred over subcutaneous administration in anaphylaxis [bib_ref] World allergy organization guidelines for the assessment and management of anaphylaxis, Simons [/bib_ref]. A unique aspect to consider in this case is the use of antihypertensive medications before the onset of the anaphylactic episode. Antihypertensive medications work on reducing BP through a wide range of different mechanisms. The antihypertensive effect usually starts as soon as the medication is administered with peak concentrations and maximal antihypertensive effects are achieved within 60 to 90 minutes depending on the type of antihypertensive agent [bib_ref] Vander Weg MW: Efficacy and safety of nighttime dosing of antihypertensives: review..., Carter [/bib_ref]. Optimal control of BP is mostly achieved a few weeks after the start and titration of antihypertensive therapy [bib_ref] How quickly should we titrate antihypertensive medication? Systematic review modelling blood pressure..., Lasserson [/bib_ref]. Chronic use of antihypertensive medications in a few studies on animal models has been shown to downregulate adrenergic receptors which can potentially contribute to reduced efficacy of IM epinephrine [bib_ref] Chronic ACE-inhibitor treatment and adrenergic mechanisms in spontaneously hypertensive rats, Castellano [/bib_ref] [bib_ref] Study of alteration of adrenergic receptor response by chronic use of lisinopril:..., Abrar [/bib_ref]. However, our patient was not on chronic antihypertensive therapy which makes it unlikely that the antihypertensive agents played a role in the reduced response to epinephrine, but they may have played a role in the drop in BP as their antihypertensive function started to take effect along with the onset of the anaphylaxis episode. In addition to epinephrine, high-flow oxygen should be given to patients with anaphylaxis. In those with laryngeal/pharyngeal edema, oxygen together with inhaled administration of epinephrine via a nebulizer is recommended. Fluid support is crucial in patients with cardiovascular involvement to help restore circulatory volume. Patients may receive inhaled beta-agonists as well as glucocorticoids to assist with bronchospasm [bib_ref] Beta2-adrenoceptors: mechanisms of action of beta2-agonists, Johnson [/bib_ref] [bib_ref] The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights, Coutinho [/bib_ref]. Despite little evidence from the literature to support the use of these drugs specifically for anaphylaxis, it is known that beta-agonists are effective in the treatment of allergic asthma and upper airway obstruction and that steroids block arachidonic acid production, thus reducing inflammation and decreasing the likelihood of protracted anaphylaxis and biphasic reactions [bib_ref] Beta2-adrenoceptors: mechanisms of action of beta2-agonists, Johnson [/bib_ref] [bib_ref] The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights, Coutinho [/bib_ref]. Systemic antihistamines have also been linked to reducing biphasic reactions, but the only established benefit is their role in relieving cutaneous symptoms [bib_ref] Beta2-adrenoceptors: mechanisms of action of beta2-agonists, Johnson [/bib_ref] [bib_ref] The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights, Coutinho [/bib_ref]. In the critical setting, the use of these drugs in a "shotgun" approach is reasonable, but further robust evidence is required to establish their role in the management of anaphylaxis. More randomized control trials and well-designed studies would significantly add to the database as there are currently limited highquality studies. Finally, it is important to educate the paramedical and medical staff on recognizing the signs and symptoms of anaphylactic shock and the management of typical and refractory cases according to the current best medical practice guidelines. # Conclusions Following evidence-based medicine is crucial in the healthcare setting. Being able to respond in a timely and effective manner to critical cases is an integral skill for any physician, especially in an emergency setting. In situations where first-line therapy is not effective, it is important to escalate management appropriately. Emergency clinicians must be able to recognize these presentations and make prompt clinical decisions. Furthermore, emergency clinicians may be faced with patients who have atypical presentations or require special consideration, such as in this case report where the patient did not respond to first-line treatments. Further research is required to establish comprehensive guidelines on the management of anaphylaxis. Currently, IM epinephrine is the mainstay treatment for anaphylaxis. IV epinephrine is not recommended as first-line but physicians with the knowledge and experience can opt to resort to it if first-line management fails. We hope this case report would add to the anaphylaxis database and demonstrate a case that has been successfully treated with IV bolus epinephrine without developing complications. # Additional information disclosures Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. [table] TABLE 1: Timeline demonstrating the interventions and corresponding vitals of the patient following administering tab amlodipine and IM diclofenac.IM: intramuscular; IV: intravenous; BP: blood pressure; HR: heart rate; BPM: beats per minute; O2: oxygen Tab captopril was administered 1 hour before administering tab amlodipine and IM diclofenac. [/table] [table] TABLE 2: Laboratory investigations following the anaphylactic episode. [/table]

In Vitro Characterization of Poly(Lactic Acid)/ Poly(Hydroxybutyrate)/ Thermoplastic Starch Blends for Tissue Engineering Application Complex in vitro characterization of a blended material based on Poly(Lactic Acid), Poly(Hydroxybutyrate), and Thermoplastic Starch (PLA/PHB/TPS) was performed in order to evaluate its potential for application in the field of tissue engineering. We focused on the biological behavior of the material as well as its mechanical and morphological properties. We also focused on the potential of the blend to be processed by the 3D printer which would allow the fabrication of the custommade scaffold. Several blends recipes were prepared and characterized. This material was then studied in the context of scaffold fabrication. Scaffold porosity, wettability, and cell-scaffold interaction were evaluated as well. MTT test and the direct contact cytotoxicity test were applied in order to evaluate the toxic potential of the blended material. Biocompatibility studies were performed on the human chondrocytes. According to our results, we assume that material had no toxic effect on the cell culture and therefore could be considered as biocompatible. Moreover, PLA/PHB/TPS blend is applicable for 3D printing. Printed scaffolds had highly porous morphology and were able to absorb water as well. In addition, cells could adhere and proliferate on the scaffold surface. We conclude that this blend has potential for scaffold engineering. # Introduction Tissue engineering (TE) is an interdisciplinary field that aims to repair various damaged tissues. Scaffolds belong to its major building blocks. They provide mechanical support for the proliferating cells and enable them to create newly formed 3D tissue by directing cell growth. For in vivo application, the material used for the scaffolding has to meet several requirements, most importantly biocompatibility and biodegradability. The importance of biodegradation is underlined with the fact that this process enables tissue remodeling. Therefore, current studies are mainly oriented on the materials, which can provide this requirement. In addition to this, proper architecture together with the physical properties facilitate cell adherence, distribution as well as differentiation into desired cell lines. When suitable polymers are applied, their degradation rate respects the regeneration process of the damaged organ so the formation of the new tissue is a regulated and balanced process. Despite the fact that various materials (both natural and synthetic origin) had been already studied in the context of TE and regenerative medicine, reconstruction of the large tissue defects still remains challenging as the sufficient vascularization for the newly engineered tissue seems to be the burning issue. Sufficient neovascularization requires a highly porous structure of the scaffold. It has been previously described that micropores facilitated ingrowth of the blood vessels and macropores influenced cell distribution and cell to cell interactions. In the presented study, we focused on the blended scaffold consisting of Poly(Lactic Acid), Poly(Hydroxybutyrate), and Thermoplastic Starch (PLA/PHB/TPS). Each polymer had been already investigated alone or as part of various blends in various medical fields (e.g., orthopedics, cardiology, neurology, urology). However, according to the best of our knowledge, no research in the field of tissue engineering and regenerative medicine has studied a blend composed of this particular formula so far. Poly (lactic acid) (PLA) belongs to a poly-lactone family and is currently considered to be the most promising component for scaffolding because of its excellent biological and mechanical properties and processability. PLA is synthesized from lactic acid, which exists in two optically active stereoisomers, L and D. Ratio of L and D form of lactic acid in PLA macromolecules has a significant effect on its resulting properties (crystalline phase volume, mechanical properties, thermal properties, degradation rate). The first step of PLA degradation is run by hydrolysis in the amorphous phase. Optically pure PLA (semicrystalline polymer) degrades slower than racemic (amorphous polymer). Choosing appropriate PLA enables to set biodegradation rate according to the needs of the tissue. However, characteristics such as degradability during processing, brittleness present PLA disadvantages. Moreover, some studies also pointed out that degradation metabolites could cause an inflammatory reaction in vivo. On the other hand, these disadvantages can be reduced or completely suppressed by combination with other polymers or by the addition of specific additives. Poly (3-hydroxybutyrate) (PHB) is a polyhydroxyalkanoate which can be produced by prokaryotic cells. Within the field of material science, its attractiveness is related to the good biocompatibility, biodegradability, and mechanical properties of this polymer. PHB is a highly crystalline polymer with a crystalline phase content from 50% to 80%. The high content of the crystalline phase makes this polymer brittle. Another disadvantage is susceptibility to thermal degradation, which complicates its thermoplastic processing. PHB degradation in vivo is a similar process as PLA degradation but lasts longer due to the high content of the crystalline phase. PHB hydrolysis metabolites are non-toxic and physiologically occur in the human body. When combined with PLA, flexible material from these brittle polymers can be engineered. Starch is a polymeric carbohydrate consisting of two different polysaccharides which are amylose and amylopectin. It had been described as a promising material for scaffold engineering thanks to its properties such as natural occurrence, biodegradability, and cost-effectiveness. Hydroxyl groups from D-glucopyranose units make starch highly hydrophilic and therefore proper biodegradation rate can be obtained. When combined with other polymers, biocompatible matrices with highly porous structure and good mechanical properties can be fabricated. For the purpose of this study, thermoplastic starch was prepared from maize starch with glycerin and water. This study is focused on in vitro complex characterization of five blends (labelled as 1-5) based on the combination of PLA, PHB, and TPS. Presented manuscript is divided into two main parts: material studies and biological studies. Within the evaluation of the material characteristics, the attention was also drawn on the possibility of tested blends to be used for 3D printing. The aim was to engineer tubular scaffolds, as repairing of the tubular tissues such as urethra or blood vessels is still challenging for the scientists. Moreover, computer aided technology is currently one of the most applied technologies within the fields of TE and regenerative medicine. The influence of the viscosity of each component on the scaffold morphology was studied as well. Other material properties included the analysis of the morphology, porosity, and ability of the material to absorb the water. The main aim of the biological studies was to evaluate biocompatibility of the tested blends as well as the cell-scaffold interactions. For the biocompatibility studies, blends were molded into thin 2D films. Cell adherence on the scaffold surface was performed on 3D planar scaffolds. # Materials and methods ## Preparation of the pla/phb/tps blend To prepare the blends, we used PLA from Nature Works LLC (Minnetonka, MN, USA) marked Ingeo Biopolymer 4060D, PHB Biomer ® batch T22 (TianAn Biopolymer, China) and TPS obtained from the company Panara (Panara Ltd., Nitra, Slovakia). We worked with three different types of TPS with different viscosity. Five different blends with different concentrations or types of TPS were tested. Blends recipes are described in. PLA/PHB ratio was 60:40. For the better mechanical properties, citrate was used as a plasticizer. Detailed recipes of the blends are under patent protection. Blends were prepared by twin screw extruder from Lab-Tech (LabTech, Bergamo, Italy) with co-rotating and fully intermeshing screws. Diameter of screws was 16 mm, L/D ratio 40. ## 3d printing of the scaffolds For the engineering of the scaffolds, we used the filament which was prepared on a single screw extruder Plasticorder Brabender (Brabedner, Duisburg, Germany) with the following parameters: L/D ¼ 25, diameter 19 mm, and screw compression ratio 1:3. The melt was extruded through a perpendicular nozzle with a diameter of 2.5 mm. In the next step, the melt was cooled by the water. The filament was processed by 3D printer Prusa I3 (Prusa Research, Prague, Czech Republic). Two types of scaffolds were prepared in the form of tubular or planar structures. The initial input data for the printing of the planar scaffolds were 3  3  0.2 cm. For the tubular scaffolds, following parameters were set: diameter 0.8 cm, wall thickness 0.2 cm and the height 2 cm. ## Viscosimetry The viscosity of the blend units was evaluated by the capillary rheometer GOTTFERT RG 20 (Göttfert, Buchen, Germany). The length of the capillary was 20 mm and it had a diameter of 2 mm. Tests were run at a temperature of 185 C. Shear rate range was from 36 to 2160 s -1 . The flow curves were corrected for non -Newtonian fluid by Rabinovich's correction. The rheological data and coefficients "n" were adjusted for the Ostwald-de Waele model. [formula] t ¼ K:g n Z ¼ dt=dg Z ¼ n:K:g nÀ1 [/formula] ## Mechanical properties of the filaments for 3d printing Tensile tests were performed on a Zwick-Roell testing machine (Zwick Roell Group, Ulm, Germany) at a crosshead speed of 50 mm/min in accordance with ISO 527. The tensile strength at break (sb) and the relative elongation at break (Eb) were evaluated from the tensile curve. ## Morphological analysis of the scaffold The microarchitecture of the tubular scaffolds was analyzed by scanning electron microscope JEOL 7500F (Jeol Ltd., Tokio, Japan). All samples had been pretreated in the boiling water for 3 hours in order to leach out the starch which served as a porogen. Samples were longitudinally broken in liquid nitrogen and brittle fractions were fixed on the mounting discs by 2-component epoxy paste. Specimens were sputter-coated with gold and platinum in an argon atmosphere for 60 s. ImageJ software was used to measure pore size. Fifty pores from each sample were randomly chosen at different sites and measured. ## Porosity The porosity of the tubular scaffolds was determined using Archimedes' principle with the ethanol used as a liquid medium. Porosity was calculated via the following equation: [formula] Porosity % ð Þ ¼ w2 À w3 À ws ð Þ =rE= w1 À w3 ð Þ =rE; [/formula] where w1 is the weight of the cylinder tube filled with ethanol, w2 is the weight of the cylinder tube containing ethanol and scaffold, w3 is the weight of cylinder tube taken out of ethanol-saturated scaffold, ws is the weight of the scaffold, and rE is the density of the ethanol. The experiment was run three times in order to obtain statistically relevant results. ## Water uptake test The swelling ratio of the tubular scaffolds was calculated via the following equation: [formula] Swelling ratio % ð Þ ¼ Ww À Wd ð Þ =Wd  100 [/formula] Firstly, all samples needed to be pretreated in the boiling water as mentioned above. Scaffolds were placed into a dry incubator for 48 hours and their dry weight (Wd) was measured on analytical weights (Kern, Frankfurt am Main, Germany). Subsequently, scaffolds were immersed in PBS and their wet weight (Ww) was measured at the predetermined time (24 hours, 2 weeks). Finally, the swelling ratio was calculated and collected data were transferred into a graph. All samples were tested in triplicates. ## Cell culture Because of the large supply of the chondral tissue, chondrocytes were chosen to be used for the further experiments. Samples were harvested in accordance with The Helsinki Declaration and isolated cells were stored in liquid nitrogen at the Institute of the Medical Biology, Genetics, and Clinical Genetics of the Comenius University (Bratislava, Slovak Republic). After the refreezing, chondrocytes were cultured in Gibco DMEM/Haḿ s F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Biosera, Nuaille, France), 1 mmol/L of glutamine (Sigma-Aldrich, St. Louis, MO, USA), 100 U/ mL of Penicillin and 100 mg/mL of Streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were plated into Petri dishes and cultured in an incubator at 37 C in a humidified atmosphere containing 5% CO 2 . After 24 hours, medium together with non-adherent cells were removed. Subsequently, the medium was changed every 3 days. After reaching 80% confluence, cells were passaged using 0.05% trypsin (Sigma-Aldrich, St. Louis, MO, USA). ## Testing of the biocompatibility Tested samples differed in the content of the TPS. Therefore, sample 1 was used because it contained the highest ratio of TPS, for the biocompatibility studies, as we had not had information about its biological behavior, which needed to be established. Cell proliferation served as an indicator of the material biocompatibility which was detected by 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and direct contact cytotoxicity test. Firstly, the MTT test was performed on chondrocytes in the co-culture with the tested material. Cells at the density of 0.4  10 5 cells per well were seeded in 24-well plates and left to adhere for 24 hours. Sample 1, in the form of 2D molded films, was cut into squares with a size of 0.5  0.5 cm. Samples were sterilized by UV light (30 minutes per each side) and immersed in a chondrocyte culture medium for 24 hours. On the next day, films were added to the cell cultures and left to affect the culture system for 24, 48, and 78 hours. After these time intervals, films together with the culture medium were removed. Subsequently, 300 mL of the fresh medium together with 30 mL of the MTT reagent (Cell Titer 96 ® AQueous One Solution Reagent, Promega, Madison, WI, USA) were added into each well. Cells were cultured at 37 C with 5% humidified CO 2 for 3 hours. ELISA microreader BioTek EL800 (BioTek Instruments Inc., Winooski, VT, USA) was used to measure the absorbance at the wavelength of 490 nm. Chondrocyte cell cultures without PLA/ PHB/Starch blend were used as positive controls. All MTT tests were performed in triplicates. Obtained data were analyzed and processed into graphs. For the direct contact cytotoxicity test, 0.6  10 5 cells per well were seeded in 24 well-plates and cultured in DMEM/ Ham's F-12 medium supplemented with 1mmol/l of glutamine, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 10% FBS at 37 C with 5% humidified CO 2 atmosphere. Sterilized 2D films, from the sample 1, were added into seeded wells and left to affect cell culture for 7 days. Cell proliferation and morphological changes were studied using an inverted light microscope (Zeiss Axiovert 100, Carl Zeiss, Jena, Germany). ## Cell seeding of the scaffolds Scanning electron microscopy (SEM) was used to evaluate the cell attachment and proliferation on the scaffold surface. According to the previous tests, sample 4 was chosen for the cell seeding experiments because of its high porosity. In brief, sterile planar 3D scaffold was placed at the bottom of the 24-well plate and seeded via droplet technique with chondrocytes. 8  10 4 cells were resuspended in 30 mL of culture medium and placed as a droplet on scaffold. After the 3 hours of the initial cell adhesion, 0.5 mL of culture medium was added into each well. Samples were cultured for 2 weeks and the medium was changed every 3 days. For the SEM analyses, sample was prepared as follow. At first, specimen was washed twice with PBS and fixed in 2.5% glutaraldehyde. Increasing concentrations of ethanol were used for dehydration and sample was subsequently left to air-dry overnight. On the next day, sample was mounted using carbon adhesive tape, sputter-coated with gold and palladium, and observed by SEM JEOL 7500F. Experiment was run in triplicate. # Statistical analysis All experiments were run in triplicates. Values were expressed as mean + standard deviation (SD). One-way ANOVA followed by a Bonferroni and Holm post hoc tests for multiple comparisons were used when appropriate. P < 0.01 and P < 0.05 were considered as statistically significant. Presented study was not blinded. # Results ## Viscosimetry In the case of polymer blending, the blend configuration preferably depends on the viscosity ratio of the blended polymers. For this reason, the rheological properties of the blends were monitored, based on these data, it is possible to predict the distribution/dispergation of TPS domains in the PLA/PHB matrix. All polymers had a pseudoplastic flow character. The individual types of TPS have sufficiently different viscosity, especially at higher shear rates. The PLA/ PHB matrix has a significantly higher viscosity than TPSs. Therefore, it is an assumption that TPS would form a continuous phase even at lower concentrations than 50% in the PLA/ PHB matrix due to the lower viscosity of TPS and at the same time, the PLA/PHB domains would be relatively large due to the big difference in the viscosities of the components. ## Mechanical properties of 3d filaments For the processability in a 3D printer, it is necessary that 3D filament has adequate mechanical properties that ensure its printability. The filament must be strong and flexible enough to ensure correct feeding and to prevent its destroying before entering to the nozzle. The continuous supply of printed material to the 3D printer's nozzle has to be ensured as well. The dependence of strength at break (sb) and the relative elongation at break (Eb) on TPS concentration was evaluated. As the TPS content increases, the tensile strength of These parameters guarantee sufficient mechanical properties of the studied 3D filament for its application in a 3D printer. ## Scaffold morphology and porosity In accordance with the previous results, we managed to engineer both planar and tubular scaffolds using computeraided technology (3D printing). When analyzed under SEM, starch proved to be a suitable porogen as we harvested highly porous structures. According to the composition of the blends, we obtained five types of scaffolds with different pore sizes, pore distribution, and orientation. Pores observed in samples 1, 3, and 4 had circular shape. When compared with samples 2 and 5, harvested images depicted more elongated pores. Pore size measurement revealed that the mean size of the pores was smaller than 10 mm. The calculated porosity of most of the structures was ! 80% except for one sample, in which calculations revealed a small porosity index. We assigned this phenomenon to the insufficient leak of the starch .. Interconnected micropores with the round shape were observed in 3 samples (1, 3, 4). Elongated pores with the different orientation were detected as well. Mean largest pore size obtained by starch leaking was 7.55 mm (3a, 3b). For the image acquisition, accelerating voltage was set to 15 kV and all samples were magnified 500 and 1000Â. ## Swelling behaviour of the scaffolds According to our results, all samples were able to absorb water while being immersed in PBS for 24 hours. When hydrated for a longer time period, only discrete water uptake was detected in 2 samples (1 and 3). What is more, weight loss was measured as well which reflected the process of biodegradation. ## Morphology of the cell culture Morphological analysis of the cell cultures intended for the biocompatibility studies was performed using SEM. Within a week after the initial seeding, chondrocytes formed a confluent colony and maintained their typical fibroblastlike shape. During the passaging, cell proliferation capacity did not decrease and no morphological changes were detected. This suggested that this cell line was stable and therefore suitable for our further experiments. Flattened cell morphology was observed when cell culture was analyzed by SEM. We could also observe nucleoli after backscattered electron detector had been used. Nucleoli are parts of the cell nucleus and their multiple occurrences indicated high proliferation activity of the chondrocytes. Passage number 10 was used in all cell tests. ## Biocompatibility studies The results of the MTT testevaluated after 24, 48, and 72 hours showed that material composition of the sample 1 did not have any significant inhibitory effect on cell proliferation. A slight decrease in the proliferation level was measured during 48 hours of the culture when compared to control group at 24 hours. However, we contributed this phenomenon to the cell adaptation process. Importantly, the proliferation capacity of the chondrocytes was up to 99% after 92 hours of the co-culture with the PLA/PHB/TPS blend. This indicated that the tested material, especially TPS, did not have a toxic effect on the cell culture.presents the result of the direct contact cytotoxicity test performed with the sample 1. After 7 days of co-culture, PLA/PHB/TPS blend (did not influence the morphology and proliferation capacity of the cells. When compared to the control group, cells maintain fibroblast-like shape and could form a compact monolayer in the co-culture with the tested material. This finding also supported the fact that no toxic metabolites had been leaked out. ## Evaluation of the cell adherence on the scaffold SEM analysis of the interference between cells and 3D planar scaffold showed that cells could attach, proliferate, and form multilayered structure on the scaffold surface, covering its entire area. The surface of the unseeded scaffold seemed to be slightly rough, which could have been caused by the presence of the TPS in the blend. Multiple filopodia, which are cytoplasmic projections of the migrating cells, underlined the successful attachment. The highest cell concentration was observed in the center of the cell-seeded construct. Cells had typical fibroblast-like morphology. Regarding the mentioned results, we assumed that the tested PLA/PHB/ TPS blend is biocompatible and therefore has the potential for the tissue engineering application. # Discussion The presented study was focused on the characterization of the blended material based on PLA/PHB/TPS in the context of scaffold fabrication. The potential of the single polymers had been already studied and the outcomes had confirmed that these biocompatible and biodegradable polymers were suitable for application in TE. Moreover, their significance is in fact that they belong to materials approved by the U.S. Food and Drug Administration (FDA) for clinical applications, such as surgical suture material and implants.However, we had not worked with TPS in the past so we needed to prove its biocompatibility along with applied plasticizers. Because of this, the sample with the highest ratio of TPS was chosen for evaluation of biocompatibility. The evaluation of the cytotoxic potential of the sample 1 according to direct contact cytotoxicity test. The control group was presented as a standard culture of chondrocytes with fibroblast-like morphology (a). PLA/PHB/TPS scaffold, which influenced cell culture for a week, did not alter cell morphology or viability, as they could form overgrown structure in the scaffolds vicinity (b, arrow depicting the edge of the 2D film). The Images were taken by inverted light microscope (Zeiss Axiovert 100, Carl Zeiss, Germany). At first, properties of the blend were assessed. Viscosity was evaluated using a capillary rheometer that simulates the flow of material through a 3D printer nozzle. It was determined that the viscosity of the individual components had a significant effect on the morphological properties of the scaffolds. It will be possible to regulate the structure of scaffolds based on PLA, PHB, and TPS according to the tissue needs by influencing the viscosity (the composition of the blend, technological parameters). TPS has a lower viscosity than PLA/PHB matrix and therefore, it is an assumption that TPS would form a continuous phase even at lower concentrations than 50%. If the concentration of TPS in the PLA/PHB matrix would be low enough to form a continuous phase it can be assumed that the TPS domains in the highly viscous matrix at sufficiently high shear rates will be small. It is possible to expect that not only the concentration of TPS but also shear rates could cause a significant change in the morphology of the scaffold. Also, slow cooling during scaffold construction can lead to the formation of clumps and larger domains. This could influence the morphology by creating larger pores. It could be possible to regulate the morphology of the scaffolds by choosing the proper processing parameters. Another goal of the material study was to evaluate the printability of the material. The prepared filaments fulfilled both physical and mechanical requirements for the 3D printing applications. The flow in the printer nozzle was continuous, there was no tearing of the filament. MTT assay and direct contact cytotoxicity test were applied in order to evaluate the biological behavior of composite material. Chondrocytes were chosen as an affected cell type for both biocompatibility tests. MTT assay was applied in order to study the instant impact of the polymers on the cell culture during short-time cultivation. We considered results obtained from the 3-day culture as the most relevant because, during the first 48 hours, the process of the cell adaptation might have influenced their vitality. Most importantly, the proliferation rate after 72 hours of coculture with the tested material was nearly 100%. This phenomenon result indicated that scaffold did not negatively affect the biological behavior of the cell culture and created a suitable environment for cells to proliferate. A recent study carried out by Ehlert et al. also described MTT assay as the test of choice for biocompatibility studies of specific alloy material. The potential toxic effect of the scaffold during long-term cultivation was analyzed by direct contact cytotoxicity test. Adverse influence might have been caused by the degradation products of the polymers. Although metabolites derived from PLA, TPS, and PHB do not present any biological risk but their altered properties might be caused by additives needed for polymer processing. These can negatively affect the pH of the culture environment and thus decrease cell viability and proliferation. According to our results, PLA/ PHB/TPS scaffold did not negatively affect surrounding cells. Moreover, cell migration from the scaffold back to the culture well was observed as well. This fact indicated that cells could attach and fully cover the surface of the matrix so extensive proliferation was present. This phenomenon was also confirmed by SEM. Morphological analysis of the scaffold revealed a highly porous structures which is the crucial property as the inner porous architecture enables effective and throughout cell incorporation. Moreover, pores also play role in neovascularization and mass transfer. The importance of the porosity was highlighted in a study carried out by Liu et al.. In this research, a cell-seeded silk fibroin/3D bladder acellular matrix was used as a composite scaffold in order to repair the complex defects of the urethral wall (animal model). High density of the macropores was achieved by electrospinning and was crucial for successful and efficient cell seeding. In our study, we attained porous architecture by starch leaching. Although we were satisfied with the overall highly porous structure, a bigger pore size would be needed. 3D printed tubular scaffolds had the biggest pore size of approximately 22 mm. However, it has been pointed out that macro pores (>100 mm) are essential for cell distribution, nutrition flow, and vascular ingrowth. On the other hand, micro pores play role in ECM formation. Ideally, scaffold should contain a mix of the micro-and a macro-pores in order to provide the best conditions for cell seeding and nutrition supply. Our pores were created only by starch leaching during pretreatment in the boiling water. The solution to this problem might be the pore size modulation through the starch domains. Then, the blend would contain both bigger and smaller starch grains which could create the desired porous structure. Another possibility presents the control of the porosity by more sophisticated 3D printing. In this case, the exact pore size and pore distribution could be obtained. Moreover, time-consuming scaffold pre-treatment could be omitted and a precise custom-made scaffold could be fabricated. The importance of 3D printing for the clinical application of tissue-engineered constructs was described in a study by Xu et al.. Results of the porosity calculation correlated with findings gathered from the electron microscopy in 4 samples. The porosity of sample 5 measured according to the Archimedes principle was only 30%. However, SEM analysis of the longitudinal sections revealed its highly porous structure. We assign this outcome to thick material layering during the 3D printing, which did not allow the effective starch leak during the pre-treating process. In this case, the modification of 3D process could solve this problem. The swelling behavior of polymers accounts for the scaffold capability to provide the required inner environment for the mass transfer, vascular supply, and cell to cell communication. Water uptake also enlarges pore size and thereby maximizes the internal surface for cell proliferation and migration. The swelling ratio of our scaffold varied from 11% to 20% during the first 24 hours. Samples with a higher concentration of TPS absorbed more water during the first 24 hours. This might have been caused by the strong hydrophobic character of TPS. When comparing the samples which contained different types of TPS, we have observed that the increasement of the viscosity was related to the lower water absorption during the first 24 hours of incubation in the water environment. This phenomenon might occur due to the used modifiers which influence the hydrophilicity of TPS. The advantage of TPS implementation into the blend is that we could modulate water absorption of the scaffold when the proper type of TPS is chosen. For further experiments, it would be useful to examine hydrophilic character of each type of TPS. A slight increasement in water adsorption was detected during scaffold incubation in the water for 2 weeks. Further analyses showed that 3 samples continued to absorb water after this time period. However, the increase was still low. After the long-time scaffold hydration, we detected that their weight started to decrease. The ratio behind this finding is probably assigned to TPS dissolution. We have also observed that the sample with the lowest content of TPS increased its weight after 3 weeks of incubation in the water -the amount of the dissolved TPS was probably lower than the amount of the absorbed water. Several studies described that water absorption should have been higher than 120%. On the other hand, excessive swelling could alter mechanical properties and degrade supporting function of the scaffold. The stiffness of the material as well as the thickness of the samples could affect our results of the water uptake. Stiffness of the scaffold needs to be altered mainly because of the potential further applications in animal models or clinical medicine because good surgical manipulation with the cell-seeded construct is essential. Scaffold was also seeded with cells in order to evaluate cell attachment and proliferation on the surface. Regarding the results from the SEM analysis, cells could interact and survive on the scaffold. Several scaffold seeding techniques had been described. We applied the droplet seeding technique. Despite we achieved successful colonization, cells were not homogenously spread and were mainly oriented in the center of the sample. Homogenous colonization can be obtained by the dynamic culture conditions. Bioreactors play a crucial role in this process and also promote tissue maturation. Moreover, this system enables the application of the physical stimuli which is crucial for the TE of the elastic and complex tissues such as blood vessels or the urethra 2,31 . The disadvantage of the bioreactor system is its cost extensiveness. Characteristic of the surface roughness plays also an important role in the cell-seeding process. Compared with the smooth surface, it had been described that scaffolds with the rough surface area were better colonized, as the overall area was bigger. In addition, spatial restriction did not allow flattening of the cells, so they maintained their shape as in a confluent population. The surface of our scaffold was smooth but starch grains could be used to modulate the roughness and create matrices with ragged topography. Another crucial step for the accurate evaluation of cellseeded construct's topography under the electron microscope is precise specimen preparation. Standard protocol involves the application of various chemicals. If handled improperly, natural shape of the cells is altered. We observed shrinkage as well as tearing of the cells in several samples after this process. Cryo-electron microscopy could offer a more sensitive approach. This method applies rapid freezing of the sample which results in the maintenance of the natural cell morphology. Moreover, cross-sections can be also performed in the vacuum atmosphere. # Conclusions We have successfully managed to develop, engineer, and characterize the PLA/PHB/TPS blend in the context of tissue engineering and regenerative medicine. Thanks to its biocompatibility and printability, we assume it is possible to apply this blend in a form of 3D scaffold in vivo. However, further studies need to be carried out in order to alter scaffold pore size and stiffness. # Ethical approval Ethical Approval is not applicable for this article. ## Statement of human and animal rights This article does not contain any studies with human or animal subjects. ## Statement of informed consent There are no human subjects in this article and informed consent is not applicable. ## Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. # Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Presented work was supported by the grant APVV-15-0111. ## Orcid id Lubos Danisovic https://orcid.org/0000-0002-5074-9621

Marginal bone loss 1 year after implantation: a systematic review for fixed and removable restorations This systematic review analyses the difference of the mean marginal bone loss (MBL) 1 year after implantation depending on the fixation of the restoration. 889 publications on controlled clinical trials were identified, and based on inclusion and exclusion criteria, 22 studies were selected. Related to fixed restorations, the lowest MBL was 0.05 ±0.67 mm and the highest 1.37±0.5 mm. The MBL for removable restorations ranged from 0.13±0.35 mm to 1.03±0.65 mm. Three studies analyzed the MBL around implants of overdentures in the lower jaw. The estimate for this restoration type was 0.476 mm (95% CI: −0.305 to 1.258). 19 randomized controlled studies dealt with restorations which were fixed to the implants. The estimate for the mean MBL was 0.459 mm (95% CI: 0.325-0.593).There was a decrease in 1-year implant survival with an increase of 1 mm MBL (−0.083%; 95% CI: −0.179 to 0.0123; p=0.083) in fixed restorations. The difference in MBL between fixed and removable restorations was 0.363 mm (95% CI: −0.319 to 1.044; p=0.279). This systematic review indicates that implants with fixed and with removable restorations lead to comparable MBL. # Introduction Edentulous patients using a conventional prosthesis suffer the loss of mastication, articulation and insufficient retention. Furthermore, this problem causes pain, loss of soft-tissue support and general dissatisfaction.Masticatory efficiency is restricted to people in possession of complete dentures, namely <20% of the masticatory performance compared to those with natural dentition. One option to overcome this issue is the use of endosseous implants.An established frequently used therapy enables the attachment of the dental implant with a denture.Van Blarcom 4 defined dental implant as A prosthetic device made of allo-plastic material(s) implanted into the oral tissues beneath the mucosal or/and periosteal layer, and on/or within the bone to provide retention and support for a fixed or removable dental prosthesis; a substance that is placed into or/and upon the jaw bone to support a fixed or removable dental prosthesis. By connecting the overdenture to the dental implant, the oral health-related quality of life (OHRQoL) improves as well as the masticatory forces increase.The removable fixation of an overdenture on two implants either splinted or unsplinted is a worldwide accepted medical treatment proven by long-term studies.Selim et al conclude in their review that the patient satisfaction of implant-supported fixed prostheses in the mandible is higher compared to the implant-supported removable overdentures. In contrast, implant-supported removable overdentures in the maxilla reach higher scores than the implant-supported fixed prostheses. The following factors are discussed: esthetics, stability, mastication performance, and pronunciation. In addition to keeping the prosthesis clean, implantsupported removable overdentures in the maxilla and mandible show favorable results.Strietzel et al check the implant loss of many different types of restorations, for example, single-tooth replacement, fixed partial denture, removable partial denture and overdenture. There is no statistically significant difference between the various types of restorations with respect to implant loss.For this reason, it is important to choose carefully which restoration is the most beneficial for the patient. Therefore, the clinician has to consider many factors before starting treatment, such as expenses, amount, arrangement and implant location, existing bone quality and quantity, maxilla-mandibular relationship, condition of the opposing dentition and time frame.To date, there is little evidence about the relation between marginal bone loss (MBL) and implant-supported fixed or removable prostheses in medical publications. This systematic review was conducted to evaluate the outcome of the mean MBL, implant and prosthesis success 1 year after implantation depending on the fixation of the restoration. # Materials and methods The present review and meta-analysis were performed according to the PRISMA guidelines.To define the research question clearly and to facilitate the process of performing the review, the PICOS approach was used. This approach is based on five components: population, interventions, comparator, outcomes and study design. ## Inclusion criteria The following study design criteria were included in the publications search: "randomised controlled study" and "follow-up one year after implantation". Criteria used to compare the test and control groups: mean age of groups, number of inserted implants, group size, loading protocol (further details see, fixed or removable restoration, implant manufacturer, treatment of implant surface (additive, subtractive, combination of additive and subtractive, combination of different subtractive treatments), survival rate based on implants and mean MBL were further requirements for inclusion. ## Exclusion criteria Exclusions to the trail were: "studies on animals or in vitro"; "reviews"; "case reports" and "clinical trials"; "follow-up one year post-loading"; and "missing data on the above-mentioned groups". ## Data extractions Two independently working reviewers (JZ and MS) extracted the data from the full text for analysis. Both reviewers double-checked the acquired information. Discrepancies were solved by mutual agreement. While reviewing the publications a chart was created and consecutively updated. The following parameters were extracted and inserted in a chart: ## Definitions In the literature, fixed restorations are described as screwed or cemented connection of the abutment to the implant body. Removable prostheses are fixed using a specific retention element to the implant. 14 # Statistical analysis In this review, language bias is non-existent, because the identified studies are written exclusively in English. Moreover, the authors tried to minimize the risk of bias by only including randomized controlled studies. Publication bias might exist because there was no access to unpublished studies. The overall MBL estimates for fixed and removable restorations were calculated using DerSimonian-Laird models random-effects meta-analysis. The Egger's test was performed to check for publication bias and the Cochran Q for heterogeneity. The association between 1-year implant survival and MBL was examined using metaregression models. The difference in meta-analytic estimates between removable and fixed restorations was tested with a metaregression including all studies using a dichotomous indicator to distinguish both restoration types. Because of the lack of information on implant success or complications in most of the studies, it was not possible to determine their relationship with MBL. Meta-analysis and metaregression were performed using STATA v.14.0 (StataCorp LP, 2015, College Station, TX, USA). # Results For creating the review, the authors used the same data referring to the searching of the three databases as shown in a previous review "Marginal bone loss one year after implantation -A systematic review for different loading protocols" .22 studies (240 implants for removable restorations and 2,096 implants for fixed restorations) were included in this review. ## Description of studies All listed studies had an observation period of 1 year after implantation using intraoral periapical radiographs.Alsabeeha et alThe estimated implant success rates for the fixed restorations range between 94.7% 33 and 100%.None of the authors mentioned the prosthesis success rate concerning fixed restorations. There are also biological and prosthetic complications listed. The most often mentioned complications concerning the biological tissue were severe MBL and periimplant mucositis. Prosthetic complications included abutment screw loosening and fracture of the restoration. Three randomized controlled clinical studies analyzed the MBL of implants which serve for better retention of overdentures. These results are illustrated in. All of them conducted examinations in the lower jaw. Alsabeeha et aldivided the patients into three equal groups: every group received a different type of implant and attachment system, but all the implants were connected to the overdenture also using the early loading protocol. Southern 8-mm-wide Implant and large ball attachments showed the best results with a measurement of MBL 0.13 mm. Neoss Regular Implants (Neoss Ltd., Harrogate, UK) and locator attachments had an MBL of 0.23 mm. The group with the Southern Regular Implants and standard ball attachments had the lowest survival rate of 90.9%, but an MBL of only 0.2 mm. The other two studies compared the immediate and early loading protocol. The groups with the immediate-loaded implants had a lower survival rate ranging between 91.7% and 93%. The MBL of overdentures loaded immediately by Maryod et alshowed a higher MBL compared to the early loaded in this study, 1.03 mm±0.61 mm versus 0.93 mm±0.52 mm. In comparison, in the study of Schincaglia et al,the results were even better, 0.25 mm±0.5 mm versus 0.54 mm±0.5 mm.shows the Forest plot on the MBL around implants supporting removable restorations a year after implantation. Theshows the Forest plot on the MBL around implants supporting fixed restorations a year after implantation. The estimate for the mean MBL was 0.459 mm (95% CI: 0.325-0.593), and heterogeneity was not significant (p=0.955). The Egger's test for freedom of publication bias had a p>0.1 (p=0.302). A decrease of −0.083% (95% CI: −0.179 to 0.013 p=0.086) in 1-year implant survival per an increase of 1 mm in MBL was observed in fixed restorations. The IQR for the 1-year implant survival reported in the considered studies was 97.0-100.0% with a median of 99.2%. The overall MBL estimates for the fixed and removable restorations did not statistically differ (0.363 mm; 95% CI: −0.319 to −1.044; p=0.279). # Discussion Patients suffering from partial or total edentulism benefit from the rehabilitation of the situation by inserting dental implants. This process shows a high satisfaction.Several prosthetic reconstructions including either fixed or removable approaches are possible.While composing this systematic review including a metaanalysis, we only searched for randomized controlled clinical trials in which the MBL was measured 1 year after implantation. Furthermore, we wanted to assess if there is a difference concerning the MBL between the two prosthetic processes. The meta-analysis showed an overall estimated MBL for the removable prostheses of 0.476 mm and for the fixed restorations of 0.459 mm. There is very little difference between these two values, which means that both prosthetic procedures lead to <0.5 mm MBL 1 year after implantation. We noticed that the randomized Regarding the studies dealing with the MBL of removable prostheses, two of three trials compared the immediate and early loading. In both controlled clinical trials, the survival rate of the immediate-loading protocol was lower. The MBL of the immediate-loading protocol measured by Schincaglia et al 17 was statistically significant (p-value <0.02) lower than the value of the early loading protocol. Comparably, Maryod et al 16 proved a statistically significant (p-value <0.011) higher MBL after 6 months of the immediate-loaded implants. But after 6 months, there was no statistically significant difference concerning MBL between the two loading protocols. To come to a decision which might be the most advantageous approach for patients in need of implant-supported overdentures, Ma et al 24 compared different loading protocols, surfaces and attachment systems for mandibular two-implant overdentures. They came to the conclusion that different attachment systems do not significantly influence the MBL. Furthermore, machined implant surfaces showed statistically significant (p-value <0.05) more MBL than subtractive methods. For the subtractive methods, they used Southern,Straumann (Straumann Group, Basel, Switzerland) and Steri-Oss (Nobel Biocare, Goteborg, Sweden) Implants. In our review, we included one study of Alsabeeha et alwhere they inserted Neoss Implants (Neoss Ltd., Harrogate, UK) between Southern Implants (Southern Implants, Irene, South Africa). Both came to similar results concerning MBL of Southern Implants. Ma et al 24 lost 0.16 mm and Alsabeeha et al 15 lost 0.13 mm in one group and 0.2 mm in the other group. MBL was statistically significantly higher (p-value <0.05) for implants loaded 2 weeks after insertion in comparison to the implants loaded 12 weeks after implantation in the study of Ma et al.The difference of MBL of implants loaded 6 or 12 weeks after implantation was not statistically significant (p-value >0.05). Concerning the implant success rate, they had comparable values to Alsabeeha et al.The measurements stayed constant after 1 year until 10 years after loading. To evaluate if there is a difference between overdentures supported by one or two implants, Tavakolizadeh et aldeveloped a study design on this topic. Twenty unsatisfied patients received either one or two interforaminal implants. After implant surgery, implants were immediately loaded. The outcome of the MBL was 0.6 mm±0.67 mm for one implant group and 0.6 mm±0.51 mm for the other. These results as well as those of Cordioli et al 26 correlate with our results. To compare this review, for the fixed prostheses, we calculated a mean MBL of 0.459 mm considering no subgroups of the fixed prostheses. The review of Firme et al 27 describes the MBL around implants supporting single fixed prostheses and multiple-unit screw-retained prostheses. They included 17 clinical trials, 7 were related to single-implant prostheses and 10 to multiple-unit screw-retained prostheses. The mean MBL and the implant success rate for the single-implant prostheses was 0.58 mm and 100%, respectively, and for the multiple-unit screw-retained prostheses the respective values were 0.9 mm and 89.1-98.9%. They showed no statistical difference (p-value >0.05) between the two types of prostheses. In this case, it has to be considered that it was not clear when the follow-up was done, 1 year after implantation or 1 year after loading. The long-term study of Lai et alshowed less MBL. The authors analyzed 231 short dental implants supporting single crowns in 168 patients using a follow-up of 1, 5 and 10 years. The MBL measured 1 year after implantation was 0.55 mm±0.45 mm. This value is comparable to our results. During the time period of 1-5 years and 5-10 years, the MBL slightly increased, with the values being 0.05 mm±0.10 mm and 0.03 mm±0.14 mm, respectively. These results indicate that most bone remodeling occurs 1 year after implantation. This systematic review and meta-analysis indicate that both the implants with fixed and with removable restorations lead to low respectively comparable MBL. However, there is a lack of clinical trials which compare these two types of restoration to each other. Further information in studies about the implant and prosthesis success rates are needed to make a clear statement. Other factors may influence the marginal bone more than the type of restoration, namely the loading protocol, or the implant surface. There is a need for further clinical trials to find the factors which lead to MBL in fixed and removable restorations supported by implants. ## Clinical, cosmetic and investigational dentistry ## Dovepress ## Publish your work in this journal Clinical, Cosmetic and Investigational Dentistry is an international, peer-reviewed, open access, online journal focusing on the latest clinical and experimental research in dentistry with specific emphasis on cosmetic interventions. Innovative developments in dental materials, techniques and devices that improve outcomes and patient satisfaction and preference will be highlighted. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. Submit your manuscript here: https://www.dovepress.com/clinical-cosmetic-and-investigational-dentistry-journal

The short and long-term effects of aerobic, strength, or mixed exercise programs on schizophrenia symptomatology The purpose of this study was to compare the effects of three different physical exercise programs on the symptomatology, body composition, physical activity, physical fitness, and quality of life of individuals with schizophrenia. A total of 432 patients were assessed for eligibility and 86 were randomized into the aerobic (n = 28), strength (n = 29) or mixed (n = 29) groups. Positive, negative, and general symptoms of psychosis, body mass index (BMI), physical activity (IPAQ-SF), physical fitness (6-min walk test [6MWT] and hand-grip strength [HGS]), and quality of life (WHOQUOL-BREF) were assessed at baseline, post-intervention (16 weeks), and at 10-months. Our results at 16 weeks showed significant improvements in all three groups in the negative, general, and total symptoms with moderate to large effect sizes (P < 0.01, η p 2 > 0.11), no change in the BMI, 6MWT or IPAQ-SF, and a significant improvement in the HGS test in the strength and mixed groups (P ≤ 0.05, η p 2 > 0.08). Nonetheless, all the improvements had disappeared at 10 months. We concluded that 3 weekly sessions of a moderate to vigorous progressive exercise program for 16 weeks improved the symptomatology of individuals with schizophrenia in all three groups, with no differences between them. However, the effects had declined to baseline levels by the 10-month follow-up, suggesting that exercise interventions should be maintained over time.Schizophrenia is a major mental health issue worldwide with a lifetime risk of approximately 0.5-1% 1 . It is a chronic disorder characterized by positive symptoms (hallucination, delusion, and thought disorders) and negative symptoms (avolition, apathy, and social dysfunction, among others) 2 . Cognitive impairment is also characteristic of these patients and appears to be related to the negative symptoms 3 , making it one of the leading causes of disability, with severe personal, social, and economic consequences 4 .A premature mortality gap of between 10 and 20 years is associated with patients with schizophrenia compared to the general population 5 , which is mainly because of premature cardiovascular disease (CVD) 6 . Moreover, genetic factors 7 and antipsychotic medications 8 strongly contribute to this high-risk profile, with unhealthy lifestyle habits such as low levels of physical activity (PA) 9 also playing a particularly prominent role in these increased mortality rates.Indeed, a recent meta-analysis demonstrated that individuals with schizophrenia engage in low levels of PA 10 and are less likely to complete PA at a sufficient intensity 11 . Accordingly, these patients tend to have low cardiorespiratory fitness 12 , which has been recognized as a strong and independent predictor of CVDs and allcause mortality in the general population 13 . In addition, lower participation in PA in these patients correlates OPEN with the presence of negative symptoms, the side-effects of antipsychotic medication, social isolation, and other unhealthy lifestyle habits 14 . Moreover, low PA may exacerbate the symptoms of depression, low self-esteem, and impair psychosocial functioning, resulting in lower health-related quality of life 15 .In contrast, exercise interventions have been consistently proven to prevent CVD 9,16 , alleviate cognitive decline 17 , and improve clinical symptoms, depression, and health-related quality of life 18 in individuals with schizophrenia. However, there are still significant gaps in the literature regarding which types of exercise might be most effective at improving symptoms, body composition, physical activity, physical fitness, and quality of life in these patients.Most work studying the effects of PA in patients suffering from schizophrenia has focused on aerobic exercise, and only a few studies have analyzed the effects of mixed (aerobic and strength) programs[19][20][21][22]. Of note, as far as we know, only two studies have used isolated strength training programs in schizophrenic patients23,24. Additionally, most of the studies have methodological limitations, which makes it difficult to draw any firm conclusions. In this context it is not surprising that several systematic reviews and/or meta-analyses of the effects of PA in patients with schizophrenia have also highlighted issues regarding methodological limitations18,25,26and claim that high-quality data from randomized controlled trials (RCTs) is still lacking.Aims of the study. The main objective of the study was to compare the immediate and long-term effects of three different physical exercise programs (strength, aerobic, or mixed) on the symptomatology, body composition, physical activity, physical fitness, and quality of life of patients suffering from schizophrenia.Materials and methodsStudy design. This prospective, multi-center, clinical trial (NCT03953664, 16/05/2019) was approved by the Ethics Committee for Biomedical Research at the University Cardenal Herrera in Valencia (Spain) and followed the ethical guidelines set out in the Declaration of Helsinki. All participants agreed to participate and signed the informed consent.Eligibility criteria. Eligible participants were all adults aged between 18 and 65 years with a diagnosis of schizophrenia (according to the DSM-5) and who were stable on antipsychotic medication, i.e., who had been using the same dosage for at least 4 weeks prior to inclusion. The exclusion criteria were: (1) a total Positive and Negative Syndrome Scale (PANSS) score of < 58 27 ; (2) acute suicidality; (3) patients representing an acute danger to others; (4) the presence of other psychiatric diagnoses or acute psychiatric illnesses; (5) other disorders that could prevent the person from completing the exercise training; (6) participation in similar programs or interventions before enrolment. Procedure. This study took place at six psychosocial care centers for people with severe mental illness (SMI), from January 2020 to January 2021. Before the start of the trial, an independent researcher unaware of the study characteristics generated a random sequence using a computerized random number generator; this was concealed from all the other study investigators throughout the entire study period. Randomization was performed with stratification for age, sex, and body mass index (BMI). Upon enrolment in the study and after completing the primary and secondary outcome measures, the participants were randomly assigned either to the aerobic (n = 28), strength (n = 29), or the mixed (n = 29) group. As shown in the participant flowchart in [fig_ref] Figure 1: Flow chart. [/fig_ref] , all the outcome measures were assessed at baseline, 16 weeks post-baseline (post-intervention), and at a 10-month follow-up. It was impossible to mask the group allocation to the physical therapists or the participants; however, the outcome evaluators and data analysts were blinded to the treatment allocations. To avoid inter-observer variability bias, the measurements in each of the groups were always completed by the same investigator. Interventions. Informed consent was obtained from the subject represented in the pictures. The intervention consisted of a total of 48 sessions (3 weekly sessions lasting 1 h each for 16 weeks). To make the comparison fair, the total number of training sessions and their duration were the same for all three training groups. The exercise training was conducted in groups at each psychosocial care center by a professional physical education instructor. Each session began and ended with 10 min of stretching of the major muscle groups. Aerobic training. Participants completed 4 series of brisk walking (in the playground of the psychosocial care centers) for 10 min followed by 1 min of recovery. To ensure that the intensity of the exercise progressed from moderate to vigorous, we monitored the heart rate (HR) of each participant using Sport testers (model 610si, Polar, Finland). Progression in exercise intensity was achieved by increasing the participant's target HR every 2 weeks. Thus, using the formula published by Tanaka et al. to calculate the maximum HR (MHR) (208-0.7 × age) [bib_ref] Age-predicted maximal heart rate revisited, Tanaka [/bib_ref] , the intensity of the exercise was progressively increased as follows: weeks 1-2: 55% MHR; weeks 3-4: 58% MHR; weeks 5-6: 61% MHR; weeks 7-8: 64% MHR; weeks 9-10: 67% MHR; weeks 11-12: 70% MHR; weeks 13-14: 73% MHR; and weeks 15-16: 76% MHR [fig_ref] Figure 2: Aerobic training program [/fig_ref]. The physical education instructor continuously checked the HR to adjust the walking speed of each participant in each session. Strength training. Participants completed two sets of 8 strength training exercises with 1 min of recovery programmed between each one [fig_ref] Figure 3: Strength training program [/fig_ref] ; informed consent was obtained from the subject represented in the picture). An elastic resistance band (Thera-band) was used in four of the eight strength exercises. The training intensity increased over the 16 weeks of this intervention; the intensity of exercises completed without an elastic band www.nature.com/scientificreports/ was amplified by increasing the number of repetitions the participants perform. For exercises performed with an elastic band, the intensity increase was achieved by using the Borg Scale. This scale measures the effort an individual perceives when exercising and creates criteria to adjust the intensity of the programmed exercise. In order to adequately use the Borg scale, the participants learnt to use Theraband resistance bands on the first day and to easily identify, for each exercise, which gripping point on the band was equivalent to an effort that was moderate, intermediate, hard or very hard according to the Borg scale. [fig_ref] Figure 3: Strength training program [/fig_ref] shows the progression (from 3 to 8) in the rate of perceived exertion (RPE) during the 16 weeks of the program. ## Follow-up (10 months) Lost to post-intervention (n = 2) (Reasons: 2 withdrew from study) Lost to post-intervention (n = 5) (Reasons: 5 withdrew from study) Lost to follow-up (n = 7) (Reasons: 5 withdrew from study, 2 were unreachable to contact) Lost to follow-up (n = 6) (Reasons: 4 withdrew from study, 2 were unreachable to contact) Lost to follow-up (n = 4) (Reasons: 3 withdrew from study, 1 was unreachable to contact) Analyzed (n = 28) Excluded from the analysis (n = 0) Analyzed (n = 29) Excluded from the analysis (n = 0) Analyzed (n = 29) Excluded from the analysis (n = 0) www.nature.com/scientificreports/ Mixed training. The main part of each mixed session was divided into two stages. First, like the strength training group, the participants performed 1 set of the same 8 strength exercises with the same intensity and progression, interspersed with 1 min of recovery time per strength exercise. Second, as in the aerobic training group, the participants performed 2 sets of brisk walking for 10 min followed by 1 min of recovery, according to the same exercise intensities and progressions described for the aerobic training group. All participants were recommended to keep a physically active lifestyle during the 10-month follow-up period. Outcome measures. Age, sex, educational level, marital status, employment, institutionalization regime, duration of illness, compliance, and adverse events to the interventions, were all registered. Antipsychotic medication (i.e., amisulpride, aripiprazole, clotiapine, clozapine, haloperidol, levomepromazine, olanzapine, paliperidone palmitate, paliperidone, quetiapine, risperidone, zuclopenthixol, and zuclopenthixol decanoate) was also recorded for each patient and were converted into a daily equivalent dosage of chlorpromazine, according to Gardner et al. [bib_ref] International consensus study of antipsychotic dosing, Gardner [/bib_ref]. In addition, anticholinergic burden (ACB) was calculated according to . Primary outcome. The PANSS is a semi-structured interview which assesses the positive (PANSS-P), negative (PANSS-N), and general (PANSS-G) symptoms of psychosis experienced by patients in the week prior on a 7-point Likert-type scale (from 1, 'none' , to 7, 'extreme') [bib_ref] The positive and negative syndrome scale (PANSS) for schizophrenia, Kay [/bib_ref]. We separately analyzed the 3 subscales as well as the overall scores (PANSS-T). Secondary outcomes. The BMI was calculated using a SECA® 780 electronic balance scale with a mechanical telescopic stadiometer. Body fat mass (BFM) was determined using a TBF-300A body-fat analyzer. A submaximal exercise test, the 6-min walk test (6MWT), was used to assess the participants' functional capacity for aerobic exercise. This test has been shown to be a reliable measure of exercise capacity in people with SMI 32 . Hand grip strength (HGS) was assessed using a Jamar hydraulic hand dynamometer, as described elsewhere [bib_ref] Reliability and validity of grip and pinch strength evaluations, Mathiowetz [/bib_ref]. The 30-s sit-to-stand (30-s STS) test was used to assess lower limb strength. It consisted of standing-up from a chair and sitting-down again as many times as possible during a 60 s period, with the researcher registering the number of repetitions [bib_ref] A 30-s chair-stand test as a measure of lower body strength in..., Jones [/bib_ref]. PA level was be assessed using the International Physical Activity Questionnaire-Short Form (IPAQ-SF) [bib_ref] Assessment of physical activity: An international perspective, Booth [/bib_ref]. Using seven items, this self-reported questionnaire collects data on the patients' PA in the 7 days prior to the test. The total number of days and minutes of PA were calculated by adding all PA category scores performed over the seven days. Data from the IPAQ-SF were converted into metabolic equivalent minutes per week (METs-min/week), using the formula published by Ainsworth et al. [bib_ref] Compendium of physical activities: A second update of codes and MET values, Ainsworth [/bib_ref]. Specifically, the IPAQ-SF questionnaire records activity at four intensity levels: (1) vigorous activity such as aerobics; (2) moderate activity such as leisure cycling; (3) walking; and (4) sitting. This makes it possible to classify the PA levels of the participants as 'high' (> 1500 METs), 'moderate' (600-1500 METs), or 'low' (< 600 METs). The IPAQ has been validated in 12 countries 37 and showed adequate psychometric properties and the short version (the IPAQ-SF) has shown acceptable validity in an adult Spanish population [bib_ref] Validity of the international physical activity questionnaire in the Catalan population (Spain), Román [/bib_ref]. Finally, quality of Life was assessed using the abbreviated WHO Quality of Life Assessment-BREF [bib_ref] Study protocol for the world Health organization project to develop a quality..., Whoqol Group [/bib_ref] : This survey comprises 26 items with five Likert-type responses each, and is a standard questionnaire used to measure patient quality of life. It assesses patients under four health domains: physical, psychological, social and environmental. In this study, we will analyze the sum of the four dimensions, with higher scores indicating a better quality of life. This scale has been validated for Spanish and the instrument has a good internal consistency with a Cronbach alpha of 0.88 for the overall scale and a range of 0.70-0.79 for its dimensions [bib_ref] Validation of the WHOQOL-BREF quality of life questionnaire among Chilean older people, Espinoza [/bib_ref]. Sample size and statistical analysis. The desired sample size was calculated after undertaking a pilot study in 15 participants 41 , which indicated a partial eta 2 effect size (η p 2 ) of 0.038 for the primary outcome (PANSS-N). Considering this and using an α value of 0.05 and a desired power of 90%, we used the G*Power 10 minutes of brisk walking. The exercise intensity should be moderate. We will control it by monitoring the heart rate (HR). Any paƟents who cannot use their HR as a reference because of their medical treatments should perform the exercise at an intensity which makes it difficult but not impossible for them to speak while performing it. ## Hr measurement See the proposed HR table created based on the paƟent age and the training program exercise week. ## Aerobic training 10 minutes of brisk walking. The exercise intensity should be moderate. We will control it by monitoring the heart rate (HR). Any paƟents who cannot use their HR as a reference because of their medical treatments should perform the exercise at an intensity which makes it difficult but not impossible for them to speak while performing it. ## Hr measurement See the proposed HR table created based on the paƟent age and the training program exercise week. ## Aerobic training 10 minutes of brisk walking. The exercise intensity should be moderate. We will control it by monitoring the heart rate (HR). Any paƟents who cannot use their HR as a reference because of their medical treatments should perform the exercise at an intensity which makes it difficult but not impossible for them to speak while performing it. ## Hr measurement See the proposed HR table created based on the paƟent age and the training program exercise week. ## Aerobic training 10 minutes of brisk walking. The exercise intensity should be moderate. We will control it by monitoring the heart rate (HR). Any paƟents who cannot use their HR as a reference because of their medical treatments should perform the exercise at an intensity which makes it difficult but not impossible for them to speak while performing it. ## Cool-down At the end of the second set: 10 mins of stretching the major muscle groups Possible confounding factors were assessed by testing baseline differences between the groups using chisquared (sex, educational level, marital status, employment, and institutionalization regime), Kruskal Wallis (age), and one-way ANOVA (illness duration, BMI, antipsychotic drugs consumption, and ACB scores) tests. ANOVA tests were also used to compare the percentage of attendance rates between the intervention groups. Two-way mixed ANOVA tests were used to compare how the study interventions affected the primary and secondary outcomes, using time (baseline, post-intervention, and 10-month follow-up) as the within-group factor and group (aerobic, strength, or mixed) as the between-group factor. Analogous ANOVA tests but with two www.nature.com/scientificreports/ levels in the time factor (baseline and 10-month follow-up) were used to compare how the study interventions affected the consumption of antipsychotic drugs. Bonferroni post-hoc tests were applied following the ANOVAs. The η p 2 effect sizes were calculated such that 0.01 < 0.06, 0.06 < 0.14, and 0.14 or higher, respectively corresponded to a small, medium, and large effect size. The statistical analyses were performed as an intention-to-treat basis using SPSS software (v.18.0) for Windows (IBM Corp., Armonk, NY). All the statistical tests were two-tailed with the critical P value for significance set at < 0.05. # Results The general characteristics of the study population are shown in . The statistical analysis did not show between-group differences at baseline for any variable, except for the number of years of illness, for which there were differences between the aerobic and mixed intervention groups. In addition, the percentages of attendance rates did not show statistically significant differences between the aerobic, strength, and mixed intervention groups (88 ± 15%, 88 ± 12%, and 79 ± 15%, respectively; P = 0.084). Likewise, the results of two-way mixed ANOVA did not show intra-group or between-group differences in the comparisons of the consumption of antipsychotic drugs. None of the participants reported any adverse events to the interventions. The results of the Bonferroni post-hoc tests are shown in . The PANSS-N, PANSS-G, and PANSS-C scores showed statistically significant improvements from baseline to 16 weeks in the 3 groups (P < 0.01, η p 2 > 0.11); however, all these significant improvements disappeared at the 10-month follow-up. The PANSS-P scores also improved in the aerobic and strength groups (P ≤ 0.005, η p 2 > 0.07), and showed a trend towards improvement in the mixed group (η p 2 > 0.04), but also got worse at the 10-month follow-up. No significant differences were shown in the intra-group comparisons of body composition (BMI and BFM), 30-s STS test, 6MWT, or IPAQ-SF. Of note, there were significant improvements in the HGS test results from baseline to 16 weeks both in the strength and mixed groups (η p 2 > 0.08). However, these improvements also disappeared at the 10-month follow-up. Similarly, the quality of life showed improvements (significantly in the aerobic group) from baseline to 16 weeks that disappeared at the 10-month follow-up. Interestingly, the intra-group comparisons between 16 weeks and the 10-month follow-up did not show statistically significant differences in symptomatology, body composition, 6MWT, or IPAQ-SF in any group. In contrast, the HGS and 30-s STS scores significantly decreased in the strength group, the HGS significantly decreased in the mixed group, and the quality of life significantly www.nature.com/scientificreports/ decreased in the aerobic group. Finally, no between-group differences were found in any of the primary or secondary outcome comparisons (P > 0.05). ## 30-s sts (repetitions) # Discussion Our results showed significant improvements in the negative, general, and overall score for the symptoms of psychosis at 16 weeks in the aerobic, strength, and mixed training groups, with moderate to large effect sizes. The mean difference in PANSS-T scores nearly reached the level of the minimal clinically important difference, estimated as a 16% to 24% change from baseline [bib_ref] What does the PANSS mean?, Leucht [/bib_ref]. These findings are consistent with several meta-analyses that showed that PA (i.e., aerobic or mixed exercise) significantly reduced the negative [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref] [bib_ref] Exercise Improves Clinical Symptoms, Quality of Life, Global Functioning, and Depression in..., Dauwan [/bib_ref] [bib_ref] Physical exercise for negative symptoms of schizophrenia: Systematic review of randomized controlled..., Sabe [/bib_ref] and total 16,18 score symptoms for schizophrenia. To date, only two studies have examined the impact of an isolated strength training program on patients with schizophrenia [bib_ref] Therapeutic effects of maximal strength training on walking efficiency in patients with..., Heggelund [/bib_ref] [bib_ref] A 20-week program of resistance or concurrent exercise improves symptoms of schizophrenia:..., Silva [/bib_ref]. The pilot study by Heggelund et al. [bib_ref] Therapeutic effects of maximal strength training on walking efficiency in patients with..., Heggelund [/bib_ref] , which consisted of an 8-week intervention (training 3 days per week) with a single exercise (4 × 4 repetitions at 85-90% of the 1 repetition maximum, performed with a leg press apparatus), did not report improvements in the negative symptoms. However, this study only included one exercise type completed for short durations, had a small sample size, and had methodological limitations (it was not randomized, assessment was not blinded, the statistical analysis did not study time × group interactions, and the data were not analyzed on an intention-to treat basis). The most recent study by Silva et al. [bib_ref] A 20-week program of resistance or concurrent exercise improves symptoms of schizophrenia:..., Silva [/bib_ref] compared the effects of 20 weeks of strength and mixed training programs (2 days per week) on psychotic symptoms. Although the authors did not find between-group differences in the PANSS-N, the intragroup analysis revealed significant improvements in the strength training group. Nevertheless, this study was also limited by some methodological issues (the sample size was small and not calculated a priori, no allocation concealment was reported, and there was no intention-to treat analysis). Therefore, our results fill a gap in literature because we consistently demonstrated that a strength training program alone was also an effective treatment for the negative and general symptoms of schizophrenia. Indeed, this type of exercise has already obtained good results in diseases such as anxiety and depression [bib_ref] Association of efficacy of resistance exercise training with depressive symptoms: Meta-analysis and..., Gordon [/bib_ref] and has been shown to increase cognitive efficiency in older adults [bib_ref] The impact of resistance exercise on the cognitive function of the elderly, Cassilhas [/bib_ref] and patients with early dementia [bib_ref] Neuropsychological and neurophysiological effects of strengthening exercise for early dementia: A pilot..., Yerokhin [/bib_ref]. Our findings showed that there were significant improvements in the positive symptoms at 16 weeks in the aerobic and strength intervention groups (with small to moderate effect sizes) and showed a positive trend in the mixed intervention group. These findings agree with other studies that analyzed the effects of aerobic interventions on positive symptoms. For example, Firth et al. [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref] conducted a meta-analysis of exercise interventions in individuals with schizophrenia and, after performing a sensitivity analysis to exclude low-intensity exercise interventions, reported significant reductions in the PANSS-P scores, with a pooled standardized mean difference (SMD) of − 0.54 (95% CI − 0.95 to − 0.13). The most recent meta-analysis [bib_ref] Physical exercise for negative symptoms of schizophrenia: Systematic review of randomized controlled..., Sabe [/bib_ref] , which included 17 RCTs, conducted a subgroup analysis that differentiated between aerobic interventions (12 RCTs) and non-aerobic interventions (5 RCTs). In this study, aerobic exercise reduced positive symptoms (SMD = − 0.27; [95% CI − 0.46 to − 0.09]; P = 0.004), while non-aerobic interventions did not reduce these symptoms (SMD = − 0.03; [95% CI − 0.29 to 0.23]; P = 0.82). Nonetheless, it must be noted that, rather than training strength, most non-aerobic intervention studies used other types of low-intensity non-aerobic exercises such as yoga [bib_ref] A randomised controlled trial of adjunctive yoga and adjunctive physical exercise training..., Bhatia [/bib_ref]. Contrary to our expectations, in this current work the intragroup analysis failed to show significant improvements at 16 weeks in the mixed training intervention group, and only showed a slight (non-significant) trend. Unlike other mixed intervention studies 21 that performed a per-protocol analysis and found significant improvements in the positive symptoms, we performed an intention-to-treat data analysis. Although the challenges of this type of analysis include possible loss to follow-up and varying compliance levels, this type of analysis maintains the original group composition achieved by randomization (avoiding potential bias due to exclusion of patients) and pragmatically estimates the benefit of the intervention by estimating how it might perform in a real clinical setting [bib_ref] Intention to treat analysis versus per protocol analysis of trial data, Sedgwick [/bib_ref]. In another vein, the fact that our results did not reach statistical significance in the mixed group could perhaps be explained both by the higher attrition rates and the lower compliance of the patients of this group. Hence, even though our 3 exercise interventions were delivered considering 2 major factors known to increase adherence and compliance in schizophrenic patients, i.e., supervision 10 and a group training setting [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref] , the percentages of dropouts were higher and the attendance rates were lower in the mixed intervention group (aerobic = 17.9% and 88%; strength = 13.8% and 88%; and mixed = 34.5% and 79%, respectively). While the mechanisms by which the different exercise interventions may have influenced the positive and negative symptoms of our patients extends beyond the scope of this study, several mechanisms have been proposed in the scientific literature. The most frequently cited are neuroprotective mechanisms such as decreased inflammation, increased neurogenesis and neuroplasticity via brain-derived neurotrophic factor, and remyelination of white matter tracts [bib_ref] Therapeutic potential of physical exercise in early psychosis, Noordsy [/bib_ref]. A recent large-scale meta-analysis confirmed that SMI patients have a significantly increased risk of CVD and CVD-related mortality and that elevated BMI in these patients, often related to antipsychotic drug use, among other factors, requires urgent clinical attention [bib_ref] Prevalence, incidence and mortality from cardiovascular disease in patients with pooled and..., Correll [/bib_ref]. In particular, weight gain induced by second-generation antipsychotic drugs is a well-documented and prevalent side effect of antipsychotic treatment [bib_ref] Antipsychotic induced weight gain: Genetics, epigenetics, and biomarkers reviewed, Shams [/bib_ref]. Thus, physical exercise is particularly recommended in this population and is included in clinical guidelines for these patients. In agreement with the literature, most of the patients in our study had baseline BMIs corresponding to excess weight (38%) or obesity (46%). In addition, all our patients were on stable antipsychotic medication, with doses that can be considered 'high' 50 corresponding to an equivalent of approximately 850 mg/day of chlorpromazine. Consistent with most previous work [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref] [bib_ref] Feasibility and effects of a group-based resistance and aerobic exercise program for..., Marzolini [/bib_ref] [bib_ref] Physical exercise keeps the brain connected: Biking increases white matter integrity in..., Svatkova [/bib_ref] [bib_ref] A 20-week program of resistance or concurrent exercise improves symptoms of schizophrenia:..., Silva [/bib_ref] , none of our three groups showed significant improvements in body composition at 16 weeks. This agrees with the finding of the meta-analysis by Firth et al. [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref] , which included studies with different types of exercise programs, that found no significant post-intervention changes in BMI www.nature.com/scientificreports/ in these patients. While achieving reductions in BMI and BFM are important exercise goals that should not be abandoned, a more realistic goal may be the attenuation of expected weight gain, to which schizophrenic patients are particularly susceptible [bib_ref] A systematic review and meta-analysis of exercise interventions in schizophrenia patients, Firth [/bib_ref]. Thus, perhaps our finding of no BMI or BFM gain should be considered noteworthy in itself. Moreover, active but obese individuals have a reduced risk for all-cause and CVD mortality compared to individuals who have a normal BMI but are physically inactive [bib_ref] Physical activity, fitness and fatness: Relations to mortality, morbidity and disease risk..., Fogelholm [/bib_ref]. Therefore, as suggested by Vancampfort et al. [bib_ref] Exercise improves cardiorespiratory fitness in people with schizophrenia: A systematic review and..., Vancampfort [/bib_ref] , exercise interventions in schizophrenic patients should be primarily developed to improve physical (cardiorespiratory) fitness, with BMI and weight reduction considered as secondary outcomes. We measured the change in functional exercise capacity using the 6MWT. This test corresponds more to the demands of everyday activities than the maximal oxygen uptake test and is less likely to evoke nervousness or anxiety [bib_ref] Reliability and intensity of the six-minute walk test in healthy elderly subjects, Kervio [/bib_ref]. Like our results, the studies by Marzolini et al. [bib_ref] Feasibility and effects of a group-based resistance and aerobic exercise program for..., Marzolini [/bib_ref] and Beebe et al. [bib_ref] Effects of exercise on mental and physical health parameters of persons with..., Beebe [/bib_ref] which implemented 12-week mixed and 16-week aerobic interventions, respectively, did not find significant improvements in the 6MWT. In contrast, Korman et al. [bib_ref] Evaluating the feasibility of a pilot exercise intervention implemented within a residential..., Korman [/bib_ref] did report a significant improvement in 6MWT results in their single-arm study (n = 10) with a 10-week mixed intervention. Of these four studies, our study started from the highest basal levels [> 560 m vs. 535 20 , 452 21 , and 430 54 ] and therefore had the lowest room for improvement. On the other hand, the systematic review by Bernat et al. [bib_ref] Six minutes walk test for individuals with schizophrenia, Bernard [/bib_ref] analyzed sixteen intervention studies and found, like our results, that none of them reported a significant increase in the distance walked by adults with schizophrenia. We measured the levels of physical activity using the IPAQ-SF. In line with the results of the 6MWT, we did not find significant changes in the levels of physical activity in any group-we only found a slight tendency towards improvement-. Although none of the exercise intervention studies discussed here [bib_ref] Evaluating the feasibility of a pilot exercise intervention implemented within a residential..., Korman [/bib_ref] [bib_ref] Feasibility and effects of a group-based resistance and aerobic exercise program for..., Marzolini [/bib_ref] [bib_ref] Physical exercise keeps the brain connected: Biking increases white matter integrity in..., Svatkova [/bib_ref] [bib_ref] Strength training restores force-generating capacity in patients with schizophrenia. Scand, Nygård [/bib_ref] [bib_ref] Therapeutic effects of maximal strength training on walking efficiency in patients with..., Heggelund [/bib_ref] [bib_ref] A 20-week program of resistance or concurrent exercise improves symptoms of schizophrenia:..., Silva [/bib_ref] [bib_ref] Effects of exercise on mental and physical health parameters of persons with..., Beebe [/bib_ref] assessed physical activity levels (IPAQ-SF) and it is not possible to make direct comparisons, a recent study suggested that the minutes of sitting reported by the IPAQ do not reflect objective sedentary behaviour measurements [bib_ref] Revisiting the International Physical Activity Questionnaire (IPAQ): Assessing sitting time among individuals..., Duncan [/bib_ref]. These authors concluded that the IPAQ may be unsuitable for the population level assessment of sitting time among individuals with schizophrenia. As expected, there were only significant intra-group improvements in strength in the strength and mixed training groups. Thus, HGS significantly increased (≥ 3 kg) in both groups. In addition, the 30-s STS test narrowly missed achieving significance in the strength group. Our results agree with other work that also found improvements in strength measured with one-repetition maximum tests after a period of isolated [bib_ref] Therapeutic effects of maximal strength training on walking efficiency in patients with..., Heggelund [/bib_ref] [bib_ref] A 20-week program of resistance or concurrent exercise improves symptoms of schizophrenia:..., Silva [/bib_ref] or mixed [bib_ref] Feasibility and effects of a group-based resistance and aerobic exercise program for..., Marzolini [/bib_ref] [bib_ref] Strength training restores force-generating capacity in patients with schizophrenia. Scand, Nygård [/bib_ref] strength training-i.e., the most recent well-designed randomized controlled trial by Nygård et al. [bib_ref] Strength training restores force-generating capacity in patients with schizophrenia. Scand, Nygård [/bib_ref] concluded that twelve weeks of maximal strength training restored patients' lower extremity force-generating capacity to a level similar to healthy references and improved 30-s STS test performance-. A cross-sectional study concluded that patients with schizophrenia showed lower HGS scores compared to healthy controls, and that HGS scores correlated positively with cognitive functions [bib_ref] Relationship of handgrip strength and body mass index with cognitive function in..., Hidese [/bib_ref]. A more recent study concluded that higher HGS was associated with greater left and right hippocampal volume and reduced white matter hyperintensities in major depressive disorder (MDD). These authors considered that interventions targeting strength fitness could improve brain health and reduce the neurocognitive abnormalities associated with MDD 58 . Moreover, HGS has been suggested as a risk indicator for cancer mortality [bib_ref] Associations of low handgrip strength with cancer mortality: A multicentre observational study, Zhuang [/bib_ref] and fatal cardiovascular and all-cause mortality events [bib_ref] Handgrip strength is inversely associated with fatal cardiovascular and all-cause mortality events, Laukkanen [/bib_ref]. Furthermore, isolated strength training has been associated with a lower risk of all-cause mortality, regardless of participation in aerobic PA [bib_ref] Strength training and all-cause, cardiovascular disease, and cancer mortality in older women:..., Kamada [/bib_ref]. To the best of our knowledge, this current study is the first RCT to compare the long-term effects of 3 different types of exercise programs on the symptomatology, body composition, physical fitness, level of physical activity, and quality of life of patients suffering from schizophrenia. Although none of the 3 programs maintained their significant effects for any variable at the 10-month follow-up, it is remarkable that, without any changes in antipsychotic medication, the variables did not worsen with respect to baseline levels. This study has limitations that must be considered. First, the patients we enrolled were (a) stable on antipsychotic medication; (b) had PANSS-T scores ≥ 58; and (c) had demonstrated an initial level of motivation to engage in the exercise programs. Therefore, our findings may only be generalizable to individuals with similar characteristics. Second, even though we based our sample size calculations on potential losses of 25%, there were 17 losses to the 10-month follow-up, which meant that our statistical analysis was underpowered by the last time point. Therefore, the long-term effects of these interventions should be interpreted with caution. Finally, the lack of a control group should be considered when interpreting the results. Controls eliminate the alternate explanations of experimental results, especially confounding variables and experimenter bias, enabling the investigator to control for threats to validity. Despite the availability of comprehensive and evidence-based treatment guidelines, not all patients benefit from standard care (e.g., medication) [bib_ref] Effectiveness of second-generation antipsychotics in patients with treatment-resistant schizophrenia: A review and..., Chakos [/bib_ref]. While anti-psychotic medication is effective for the positive symptoms of schizophrenia [bib_ref] Comparative efficacy and tolerability of 15 antipsychotic drugs in schizophrenia: A multiple-treatments..., Leucht [/bib_ref] , it is less effective for the treatment of its negative symptoms and cognitive deficits 64 . Unfortunately, these deficits constitute major determinants of poor functioning and disability 3 and negative symptoms are an important predictor of an unfavorable disease course [bib_ref] Physical exercise for negative symptoms of schizophrenia: Systematic review of randomized controlled..., Sabe [/bib_ref]. Thus, adjunctive therapies like physical exercise that can reduce negative symptoms, cognitive deficits, and improve functional outcomes are vital. Perspective. This study concluded that 3 weekly sessions of a moderate to vigorous progressive exercise program for 16 weeks improved the symptomatology of schizophrenic patients in all three groups (aerobic, strength, or mixed), with no differences between them. Additionally, the greatest improvements in strength were achieved with interventions that included strength training exercises. However, their positive effects declined to baseline levels at the 10-month follow-up, suggesting that PA programs should be maintained for longer. Future studies should be deigned to elucidate strategies to keep patients with schizophrenia physically active over time. ## Data availability Data available on request from the authors. [fig] Figure 1: Flow chart. [/fig] [fig] Figure 2: Aerobic training program. [/fig] [fig] Figure 3: Strength training program. [/fig] [fig] Table 1: Baseline participant characteristics. BMI body mass index, BFM body fat mass, PANSS positive and negative syndrome scale, HGS hand-grip strength, 30-s STS 30-s sit-to-stand test, 6MWT 6-min walk test, IPAQ-SF International Physical Activity Questionnaire-Short Form, MET metabolic equivalent of task, WHOQUOL World Health Organization Quality of Life. [/fig]

Lost in translation—the use of remote and on-animal sensing for extensive livestock systems # Introduction Remote and on-animal sensors underpin the development of Precision Livestock Farming (PLF). These technologies have been demonstrated in intensive livestock production resulting in decreased labor costs and in the establishment of animal production as an industrial process. This level of process control is not appropriate for extensive, large-scale pastoral, and mixed farm grazing. By definition, this is an industry sector where manual inputs must be minimized, the interaction between livestock and the environment is a priority, and management flexibility is essential. Nevertheless, the opportunity to monitor animals remotely has many attractions and it is appropriate to review how research and development over the past 20 yr have progressed toward application. ## Technology for cropping The use of geographical positioning systems (GPS), geographical information systems (GIS), remote sensing, and other information technology is now widespread in broadacre cropping. This cropping can be carried out during short but intensive activity periods using large machinery. The technology allows precise applications of fertilizer, seed, and herbicide best suited for specific soils and topography. The machinery does not need to be driven but can be monitored from within an airconditioned cab or even from a remote location. Technological development is progressing rapidly with options such as the use of unmanned aerial vehicles (UAVs) for spraying and crop monitoring close to application. This application and adoption demonstrate that there are appropriate technical skills in the rural sector and a willingness of progressive farmers to adopt change and new technology. Parallel changes have not occurred in the grazing livestock industry suggesting that appropriate technology packages are not available. ## Remote and on-animal sensors for livestock The potential for the use of remote and on-animal sensing to improve the production and welfare of grazing livestock and enhance landscape management is transformational. High-value quantitative information will enable a step change in the precision management of pastures and grazing sheep and cattle. Opportunities for remote monitoring and management include pasture growth rates and feed on offer from satellite imagery, walk over weighing, virtual fencing, remote drafting for supplement management, UAVs for tracking and control, and the use of cameras or sensors for monitoring water points. Pasture monitoring provides an indicator of available feed in a constantly changing and complex mosaic of pasture phenologies. Virtual fencing, remote drafting, gate management, and application of UAVs for movement control may support active management not simply monitoring of activity. On or in-animal sensorscan be used to measure a range of different behaviors and characteristics such as distances walked, direction, velocity, acceleration, posture, location, proximity to other animals, body movements (e.g., jaw and bite), body temperature, heart rate, and Implications - Remote and on-animal technology has been used successfully for intensive animal production where process control is possible. Although grazing livestock systems are different, there is vast potential to use similar technology for industry transformation. - Despite this, technology adoption has been slow and the transformations predicted 10-15 years ago have not eventuated. - The current paradigm is not working implying a change is required. It is advocated that a new research model based on requirements for technological packages, designed through a consultative process involving farmers, adoption specialists, engineers and scientists be used to set priorities for future research. mounting activity. The collected data have then been related to various indicators of health, production, or welfare such as heat stress and water requirements, metabolizable energy intake, parasite infection, dog or wolf predation, estrus detection (Alhamada et al., 2016), parturition time, and dam parentage. There is potential for much more; for example, a recent publication describing the relationship between observed behavior and flystrike is a candidate for an on-animal sensor, and the relationship between time spent grazing, feed availability, and quality and ground coveralso has great potential but has not been adequately investigated. In Australia, where one person (with the help of occasional contractors) may manage 5,000 to 10,000 sheep spread across many paddocks and flocks, collection of even a small proportion of the stream of information available from remote sensing through manual measurement and observation would be impossible. Similar conclusions apply to cattle grazing rangelands and extensive pastures. This means that the effective use of remote and on-animal sensors would lead to improvements in efficiency of feed use, environmental management of the farm landscape, welfare, and production and not just to reduced labor costs. ## Adoption and utilization Despite the demonstrated capabilities of the technologies, application to facilitate remote PLF has been almost nonexistent. Predictions made in 2012 that PLF would revolutionize the livestock industry in the next 10 yr and that sensors would be deployed routinely around animals to allow farmers to effectively monitor a range of useful parametershave not eventuated. Why has progress has been so slow? This question has been partly addressed in a review by, in which the authors state "applications are mostly based on a single or limited number of indicators or sensors, rather than the desired large number of indicators, which would improve quality of the application." This is clearly true as 68 of the 71 peerreviewed articles listed bydescribed the use of either one or two on-animal sensors; moreover, results are usually presented in the context of the individual animals rather than flocks or whole farm.go further and identify the lack of interoperability of various applications as a major challenge. All of this indicates that end users of the technology are not being supplied with solutions. Complex data flows from multiple sources are not useful; farmers will want to turn on a computer or phone at a remote location and be provided with integrated information on feed and water availability, animal weight and condition, and potential health and welfare to support day-to-day management activities. ## Future research and development It is time to reset the research direction and priorities on the use of technology for grazing systems. There are three clear priorities that can be derived from gaps in published research. The first priority is to establish end user interest and direction. Without enthusiastic support, direction, and commitment by livestock managers, the technology is likely to stall. Only through a well-organized industry consultation process can an end product(s) be designed that will have a chance of adoption. This should include the definition of the questions and solutions that livestock managers consider a priority, reviewed in the context of what technology can deliver, and how it could be packaged. This will also inform the process of sensor aggregation to measure multiple parameters. Very similar comments were made by others 13 yr agobut appear to have been ignored. After consultation, the second priority is integration and intelligent analysis of the collected data to meet design requirements. For example, plentiful pasture accompanied by low intake and poor growth could contribute to the interpretation of behavior change consistent with flystrike, parasites, or nutrient imbalance. This type of conclusion requires the interpretation of multiple data sources. Finally when the product(s) have been designed and a logical framework for data analysis and interpretation completed, the capability to share relevant data across different hardware and applications (interoperability)will be needed. This process should be demand driven. In reviewing future research and development directions, it is clear that progress will only be made through the formation and support of multidisciplinary teams of engineers, livestock and pasture scientists, adoption experts, and end users. ## Literature cited ## About the authors ## David masters is an adjunct Professor at the University of Western Australia in the School of Agriculture and Environment and an Honorary Fellow with CSIRO. He has a background in nutritional physiology and biochemistry with a specific interest in mineral metabolism. During his 30 yr research career, he has channeled his scientific background into research on applied livestock systems and particularly into managing grazing livestock for positive environmental outcomes. He initiated national programs to reduce the impact of salinity in Australian agriculture through the use of grazing plants and has published on both greenhouse gas from grazing livestock and remote monitoring of movement and behavior. Recent reviews cover topics such as halophytes in grazing systems, physiological changes associated with genetic selection, dystocia in sheep, and the practical implication of mineral deficiencies in grazing livestock. Corresponding author: [email protected]

Renal Hypoxia in CKD; Pathophysiology and Detecting Methods Chronic kidney disease (CKD) is a major public health problem. Accumulating evidence suggests that CKD aggravates renal hypoxia, and in turn, renal hypoxia accelerates CKD progression. To eliminate this vicious cycle, hypoxia-related therapies, such as hypoxia-inducible factor (HIF) activation (prolyl hydroxylase domain inhibition) or NF-E2-related factor 2 activation, are currently under investigation. Clinical studies have revealed heterogeneity in renal oxygenation; therefore, the detection of patients with more hypoxic kidneys can be used to identify likely responders to hypoxia-oriented therapies. In this review, we provide a detailed description of current hypoxia detection methods. HIF degradation correlates with the intracellular oxygen concentration; thus, methods that can detect intracellular oxygen tension changes are desirable. The use of a microelectrode is a classical technique that is superior in quantitative performance; however, its high invasiveness and the fact that it reflects the extracellular oxygen tension are disadvantages. Pimonidazole protein adduct immunohistochemistry and HIF activation detection reflect intracellular oxygen tension, but these techniques yield qualitative data. Blood oxygen level-dependent magnetic resonance imaging has the advantage of low invasiveness, high quantitative performance, and application in clinical use, but its biggest disadvantage is that it measures only deoxyhemoglobin concentrations. Phosphorescence lifetime measurement is a relatively novel in vivo oxygen sensing technique that has the advantage of being quantitative; however, it has several disadvantages, such as toxicity of the phosphorescent dye and the inability to assess deeper tissues. Understanding the advantages and disadvantages of these hypoxia detection methods will help researchers precisely assess renal hypoxia and develop new therapeutics against renal hypoxia-associated CKD. # Introduction Chronic kidney disease (CKD) remains a major public health problem in both developed and developing countries despite enormous treatment efforts [bib_ref] United states renal data system public health surveillance of chronic kidney disease..., Collins [/bib_ref] [bib_ref] Worldwide access to treatment for end-stage kidney disease: a systematic review, Liyanage [/bib_ref]. Many clinical conditions, such as diabetes mellitus, glomerulonephritis, hypertension, and genetic disorders, are causative factors of CKD. However, once renal fibrosis, a major pathological hallmark of CKD, reaches a certain threshold, CKD progression becomes irreversible and independent of the initial cause [bib_ref] Is there a common mechanism for the progression of different types of..., Fine [/bib_ref] [bib_ref] Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal..., Nangaku [/bib_ref]. Thus, the final common pathways of CKD progression have been extensively studied. Renal hypoxia (i.e., decreased oxygen tension in the kidney), particularly in the tubulointerstitium, is a candidate prognostic marker for CKD progression [bib_ref] Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal..., Nangaku [/bib_ref] [bib_ref] The suffocating kidney: tubulointerstitial hypoxia in end-stage renal disease, Mimura [/bib_ref]. Increased hemoglobin concentrations, which ameliorate renal hypoxia, are associated with a better renal prognosis in CKD patients [bib_ref] High target hemoglobin with erythropoiesis-stimulating agents has advantages in the renal function..., Tsubakihara [/bib_ref]. In support of hypoxia as a marker of poor renal function, sleep apnea syndrome and chronic obstructive pulmonary disease have been identified as risk factors for CKD [bib_ref] High Prevalence of chronic kidney disease among patients with sleep related breathing..., Iseki [/bib_ref] [bib_ref] Sleep apnea and the kidney: is sleep apnea a risk factor for..., Hanly [/bib_ref] [bib_ref] Chronic obstructive pulmonary disease is associated with risk of chronic kidney disease:..., Chen [/bib_ref]. Although these studies are observational and no intervention studies with domiciliary oxygen therapy or continuous positive air pressure ventilation have been conducted, our results raise the possibility that conditions, such as SAS and COPD result in intermittent or continuous hypoxemia and increase the risk of CKD development via renal hypoxia aggravation. ## Cellular response to hypoxia Oxygen is imperative for mitochondrial oxidative phosphorylation, a process that produces sufficient ATP amounts in metazoans. Thus, cellular response against insufficient oxygen supply is precisely controlled. Transcriptional factors known as hypoxia-inducible factors (HIFs) play a central role in the antihypoxia defense system [bib_ref] Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology, Semenza [/bib_ref]. HIFs are composed of two subunits: HIF-α, an oxygen-sensitive subunit, and HIF-β, an oxygen-insensitive subunit. The cellular concentration of HIF-α is strictly tuned by the oxygen tension. Prolyl hydroxylase domain (PHD) proteins hydroxylate HIF-α using oxygen molecules as substrates. Next, hydroxylated HIF-α recruits von Hippel-Lindau tumor suppressor, which triggers ubiquitination and proteasomal degradation . In this mechanism, HIF-α hydroxylation is the rate-limiting step. Therefore, the cellular concentration of HIF-α is dependent on oxygen tension. Given that HIF-α hydroxylation occurs in the cytosol, the intracellular oxygen tension is more important than the extracellular oxygen tension, which is usually estimated by measuring the intravascular oxygen tension. Increases in cytoplasmic HIF-α concentrations due to decreased intracellular oxygen tension or PHD pharmacological inhibition lead to the formation of a heterodimer between HIF-α and HIF-1β. The heterodimer translocates into the nucleus and binds to the consensus enhancer via hypoxia-responsive elements (HREs), which then upregulates downstream target genes. The representative downstream genes include vascular endothelial growth factor (VEGF), erythropoietin (EPO), glucose transporter 1, and heme oxygenase 1, which protect the cell from hypoxia by promoting neovascularization, erythropoiesis, and glycolysis and by decreasing oxidative stress, respectively. There are three isoforms of HIF-α: HIF-1α, HIF-2α, and HIF-3α. In the kidney, HIF-1α is dominantly expressed in tubular cells, whereas HIF-2α accumulates in interstitial and endothelial cells [bib_ref] Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys, Rosenberger [/bib_ref]. The expression and function of HIF-3α remains elusive. Full-length HIF-3α works as a transcriptional factor with HIF-1β; however, some of its splice variants work as dominant negative forms against HIF-1α and HIF-2α [bib_ref] Hypoxia-inducible factor 3 biology: complexities and emerging themes, Duan [/bib_ref]. ## Renal hypoxia pathophysiology The oxygen tension in the kidney is relatively lower in healthy individuals, even though the kidneys receive approximately 20% of the blood pumped out from the heart. One explanation is the oxygen shunt between the arterial and venous vessels that run parallel in the kidney, as proven by oxygen electrodes [bib_ref] Evidence for a preglomerular oxygen diffusion shunt in rat renal cortex, Schurek [/bib_ref]. Recently, it has been actively debated whether an arteriovenous oxygen shunt affects renal oxygenation based on results from a three-dimensional computational model. To obtain a clear answer, the quantification of the geometry of artery-vein pairs in the kidney may be needed as results from the three-dimensional computational model vary with the assumption of background vascular networks [bib_ref] Vascular geometry and oxygen diffusion in the vicinity of arteryvein pairs in..., Ngo [/bib_ref] [bib_ref] The plausibility of arterial-to-venous oxygen shunting in the kidney: it all depends..., Evans [/bib_ref] [bib_ref] Renal oxygenation: preglomerular vasculature is an unlikely contributor to renal oxygen shunting, Olgac [/bib_ref]. An oxygen shunt supposedly affects renal oxygenation and is likely accountable for the higher oxygen tension in renal veins than in efferent arterioles [bib_ref] Nephron pO 2 and renal oxygen usage in the hypertensive rat kidney, Welch [/bib_ref] and for the oxygen gradient in the kidney, i.e., the oxygen tension decreases deeper in the renal surface. The oxygen tension in the renal surface is approximately 50 mmHg, whereas the tension in the renal medulla is approximately 30 mmHg, as detected by microelectrodes [bib_ref] Kidney hypoxia, attributable to increased oxygen consumption, induces nephropathy independently of hyperglycemia..., Friederich-Persson [/bib_ref] [bib_ref] Determinants of renal tissue hypoxia in a rat model of polycystic kidney..., Ow [/bib_ref] [bib_ref] Activation of hypoxia-inducible factors prevents diabetic nephropathy, Nordquist [/bib_ref]. A lower oxygen tension in normal kidneys has also been shown in a study that visualized HIF activity in transgenic mice [bib_ref] Mouse model for non-invasive imaging of HIF prolyl hydroxylase activity: assessment of..., Safran [/bib_ref]. In these mice, the luciferase enzyme was fused to the oxygen-dependent degradation domain of HIF-1α. Thus, luciferase activity simulated HIF-α expression. Under normoxic conditions, the kidneys were the only organ that expressed sufficient luminescence that could be observed in the in vivo system. In addition to its innate low oxygen tension, the kidneys suffer from severe hypoxia as CKD progresses. Renal hypoxia pathophysiology in CKD includes a decrease in oxygen supply and an increase in oxygen consumption. Loss of peritubular capillaries (PTCs) [bib_ref] Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular..., Kang [/bib_ref] [bib_ref] Peritubular capillary regression during the progression of experimental obstructive nephropathy, Ohashi [/bib_ref] , reduction in PTC flow , and renal anemia [bib_ref] Association of kidney function with anemia: the third national health and nutrition..., Astor [/bib_ref] account for insufficient oxygen supply. Increases in oxygen demand in renal tubules have been shown in a reduced renal mass model [bib_ref] Remnant kidney hypermetabolism and progression of chronic renal failure, Harris [/bib_ref] [bib_ref] Dietary deficiency of antioxidants exacerbates ischemic injury in the rat kidney, Nath [/bib_ref] , hypertensive model [bib_ref] Impaired regulation of renal oxygen consumption in spontaneously hypertensive rats, Adler [/bib_ref] , and diabetes mellitus model [bib_ref] Increased renal metabolism in diabetes. Mechanism and functional implications, Korner [/bib_ref] , and this increase in the reduced renal mass model is corrected by renin-angiotensin-aldosterone axis blockade or HIF activation [bib_ref] Regulation of oxygen utilization by angiotensin II in chronic kidney disease, Deng [/bib_ref] [bib_ref] Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II..., Deng [/bib_ref]. These hypoxic changes likely exacerbate renal tubular damage. Outer medullary areas, including the S3 segment of the proximal tubules, medullary thick ascending limbs, and medullary collecting ducts, are considered to be most susceptible to hypoxia because FIGURE 1 | HIF-α regulation. In the presence of sufficient oxygen, HIF-α is hydroxylated by PHD. Hydroxylated HIF-α is recognized by vHL, which results in proteasomal degradation. In hypoxic conditions or PHD inhibition, HIF-α accumulates in the cytosol and forms a heterodimer with HIF-β, the hypoxia-insensitive unit. The heterodimer translocates to the nucleus and acts as a transcriptional factor that binds to HREs. Abbreviations: HIF, hypoxia-inducible factor; HREs, hypoxia-responsive elements; PHD, prolyl hydroxylase domain; vHL, von Hippel-Lindau disease tumor suppressor. histological changes are obvious in these segments of human kidneys with ischemia [bib_ref] Experimental ischemiareperfusion: biases and myths-the proximal vs. distal hypoxic tubular injury debate..., Heyman [/bib_ref]. Differences in several features, including mitochondrial density and transport activity, account for the susceptibility to hypoxic insults. The medullary thick ascending limbs and medullary collecting ducts become more susceptible to hypoxic insults with forced tubular transport [bib_ref] Transport-dependent cell injury in the S3 segment of the proximal tubule, Shanley [/bib_ref] [bib_ref] Hypoxia of the renal medulla-its implications for disease, Brezis [/bib_ref] [bib_ref] Oxygen and renal metabolism, Epstein [/bib_ref]. ## Hypoxia-oriented therapy against ckd Renal hypoxia accelerates CKD progression, and in turn, CKD exacerbates renal hypoxia. Therefore, eliminating this cycle is a promising strategy against CKD. One approach is HIF activation via PHD inhibition. Cobalt chloride is a classical PHD inhibitor, and it ameliorates CKD progression in a model of STZ-induced type 1 diabetes and Thy-1 nephritis and a remnant kidney model [bib_ref] Activation of hypoxia-inducible factors prevents diabetic nephropathy, Nordquist [/bib_ref]. However, this treatment cannot be used in CKD patients because of severe adverse effects. PHD inhibitors that specifically inhibit HIF-α hydroxylation have recently been synthesized. These compounds are currently in clinical trials for renal anemia because the EPO gene is markedly upregulated by HIF-α stabilization [bib_ref] Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory..., Fraisl [/bib_ref] [bib_ref] HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond, Maxwell [/bib_ref] , and several drugs show positive outcomes in clinical trials in non-dialysis CKD patients and dialysis patients [bib_ref] A novel hypoxia-inducible factor-prolyl hydroxylase inhibitor (GSK1278863) for anemia in CKD: a..., Brigandi [/bib_ref] [bib_ref] Four-Week studies of oral hypoxia-inducible factor-prolyl hydroxylase inhibitor GSK1278863 for treatment of..., Holdstock [/bib_ref] [bib_ref] Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in non-dialysis-dependent..., Pergola [/bib_ref] [bib_ref] Oral hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat (FG-4592) for the treatment of..., Provenzano [/bib_ref]. These drugs are expected to have protective effects against CKD. ## Oxidative stress-oriented therapy against ckd Another treatment approach is the enhancement of the antioxidant response because hypoxia is closely related to increased oxidative stress. One relevant transcriptional factor for this approach is NF-E2 related factor 2 (Nrf2). Nrf2 is degraded via recognition by Kelch-like ECH-associated protein 1 (Keap1) in the cytosol under normal conditions. When certain chemical stresses, such as oxidative stress, are induced, Nrf2 escapes degradation, translocates into the nucleus, binds to anti-oxidant responsive elements (AREs), and upregulates downstream genes [bib_ref] The Nrf2 cell defence pathway: keap1-dependent and -independent mechanisms of regulation, Bryan [/bib_ref] ; . Nrf2-ARE axis activation induces strong anti-oxidative effects; therefore, the pharmacological activation of the Nrf2-ARE pathway is a promising target for various diseases, including kidney diseases. Bardoxolone methyl, an Nrf2 activator via Keap1 inhibition, increases eGFR in CKD patients with diabetes mellitus; however, the change in eGFR is not considered primary or secondary outcomes, and hypertension and an increased urinary albumin-to-creatinine ratio was seen in the bardoxolone group [bib_ref] Bardoxolone methyl and kidney function in CKD with type 2 diabetes, Pergola [/bib_ref] [bib_ref] Bardoxolone methyl in type 2 diabetes and stage 4 chronic kidney disease, De Zeeuw [/bib_ref]. However, a high frequency of cardiovascular events in the bardoxolone methyl group led to the termination of the phase III trial [bib_ref] Bardoxolone methyl in type 2 diabetes and stage 4 chronic kidney disease, De Zeeuw [/bib_ref]. A subsequent analysis revealed that most of these patients suffered from excessive fluid retention at the early stage. Considering that these clinical signs can be predicted under close follow-up of the patients and that diabetic kidney disease is major cause of end-stage renal disease, the potential benefit of this drug was reconsidered and a new phase II trial that excludes patients with a high risk of cardiovascular disease is ongoing in Japan. FIGURE 2 | Nrf2 regulation. Under normal conditions, Nrf2 is recognized by KEAP1, which triggers its proteasomal degradation. In contrast, under stressful conditions, such as increased oxidative stress, Nrf2 recognition by KEAP1 is disturbed, and Nrf2 accumulates in the cytosol and acts as a transcriptional factor via ARE binding. Abbreviations: ARE, anti-oxidant responsive element; KEAP1, Kelch-like ECH-associated protein 1; Nrf2, NF-E2 related factor 2. These hypoxia-targeting pharmaceutical therapies are promising. Theoretically, these therapies are effective only on patients whose kidneys are truly hypoxic. The renal oxygen state widely varies in CKD patients [bib_ref] Renal BOLD-MRI does not reflect renal function in chronic kidney disease, Michaely [/bib_ref]. Thus, it is important to identify patients whose kidneys are sufficiently hypoxic, i.e., more responsive to hypoxia-oriented therapies. However, the currently available hypoxia detection methods have serious disadvantages. Thus, there is a compelling need for a better understanding of hypoxia detection methods. ## General principles of oxygen tension assessment The most important function of oxygen molecules is their action as an oxidant. In addition, oxygen can bind to hemoglobin to increase its solubility in blood. A lower oxygen tension results in HIF-HRE axis activation. Thus, we consider that methods to measure the oxygen tension can be divided into four groups: (i) assessment of the oxidative power of oxygen molecules [bib_ref] Monitor and control of blood and tissue oxygen tensions, Clark [/bib_ref] [bib_ref] Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde..., Arteel [/bib_ref] , (ii) measurement of the oxyhemoglobin/total hemoglobin ratio [bib_ref] Non-invasive evaluation of intrarenal oxygenation with BOLD MRI, Prasad [/bib_ref] , (iii) detection of HIF activity [bib_ref] Mouse model for non-invasive imaging of HIF prolyl hydroxylase activity: assessment of..., Safran [/bib_ref] , and (iv) direct measurement of oxygen molecules by the assessment of fluorescence or phosphorescence quenching rate [bib_ref] Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused..., Rumsey [/bib_ref] [bib_ref] Mitochondrial PO 2 measured by delayed fluorescence of endogenous protoporphyrin IX, Mik [/bib_ref]. Although these four methods unequivocally depend on the oxygen tension, there are some confounding factors in their interpretation. For example, when the oxidative power of oxygen molecules is measured, other oxidants or reductants can influence the result. Notably, oxidative stress levels increase in CKD patients [bib_ref] The elephant in uremia: oxidant stress as a unifying concept of cardiovascular..., Himmelfarb [/bib_ref]. Although the oxyhemoglobin/total hemoglobin ratio is clinically used to determine a patient's general oxygenation state, this value can be affected by the partial pressures of oxygen and carbon dioxide, changes in pH, and other micro-environmental changes (Bohr effect). Blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) employs hemoglobin as an oxygen sensor; however, this method only measures the deoxyhemoglobin concentration and not the deoxyhemoglobin/total hemoglobin ratio. Thus, this method is influenced by the hemoglobin concentration, which cannot be precisely measured in small veins [bib_ref] Non-invasive evaluation of intrarenal oxygenation with BOLD MRI, Prasad [/bib_ref]. Renal anemia and acid-base disorders frequently occur in CKD patients, which may explain why the use of renal BOLD-MRI in CKD patients remains controversial [bib_ref] Non-invasive evaluation of kidney hypoxia and fibrosis using magnetic resonance imaging, Inoue [/bib_ref] [bib_ref] Renal BOLD-MRI does not reflect renal function in chronic kidney disease, Michaely [/bib_ref] [bib_ref] Determinants of renal tissue oxygenation as measured with BOLD-MRI in chronic kidney..., Pruijm [/bib_ref]. HIF activation may be influenced by factors other than oxygen molecules, such as erythropoietin and indoxyl sulfate [bib_ref] Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent..., Chiang [/bib_ref]. Indoxyl sulfate, a representative uremic toxin, suppresses EPO gene transcription in hypoxic HepG2 cells in an HIF-dependent manner, and the oral administration of indole, a precursor of indoxyl sulfate, decreases the serum erythropoietin concentration in rats. Another important consideration is whether the method employed reflects the intracellular or extracellular oxygen tension. As previously noted, the HIF-mediated cellular response against hypoxia is regulated by the hydroxylation of HIF-α, which uses oxygen as a substrate. Thus, the intracellular oxygen tension is important. For example, anemia can cause dissociation between the intracellular and extracellular oxygen tensions because anemia does not change the partial pressure of oxygen, but rather changes the total oxygen amount in arterial blood (i.e., decrease in hemoglobin concentration). This condition should not be overlooked because renal anemia development is associated with renal hypoxia in CKD patients. Another important aspect to consider is the generation of quantitative or qualitative data. Microelectrodes, BOLD-MRI, pimonidazole protein adduct immunohistochemistry, and HIF activation detection are frequently used as hypoxia detection methods [bib_ref] Renal parenchymal hypoxia, hypoxia response and the progression of chronic kidney disease, Heyman [/bib_ref]. Phosphorescence lifetime measurement is a relatively novel intravital oxygen sensing technique; thus, we have included this technique in our review [bib_ref] Imaging local neuronal activity by monitoring PO 2 transients in capillaries, Parpaleix [/bib_ref] [bib_ref] Direct measurement of local oxygen concentration in the bone marrow of live..., Spencer [/bib_ref] [bib_ref] Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement, Hirakawa [/bib_ref]. The characteristics of these methods are summarized in [fig_ref] TABLE 1 |: Comparison of hypoxia detection methods [/fig_ref]. For clinical applications, BOLD-MRI is the only appropriate method. For animal experiments, microelectrodes are a gold standard for oxygen tension measurement, although the specific measurement site is unclear. Pimonidazole staining and HIF activation detection can be used for paraffin sections and has the advantage of spatial resolution. Phosphorescence lifetime measurement is a highly sensitive method, but it requires special instrumentation. Comparison of results obtained via different methods is attractive, and there is a report that compared pO 2 measured by a microelectrode with that measured by a fluorescence needle probe in the kidney and which mentions that there is a slight difference in outcomes between these two methods [bib_ref] Renal medullary tissue oxygenation is dependent on both cortical and medullary blood..., O&apos;connor [/bib_ref]. However, this comparison cannot determine which of the two methods is reliable; the calibration method of each method differs, and the measurement site is not always the same. Therefore, the comparison of pO 2 obtained via different techniques needs to be interpreted with that consideration in mind. # Hypoxia detection methods ## Microelectrodes The use of microelectrodes is a classical technique and the gold standard for determining the oxygen tension in living cells and animals. The principle of this technique is based on oxidationreduction reactions. The reaction for a silver electrode is shown below: [formula] 4Ag + 4Cl − → 4AgCl + 4e − · · ·(1)4H + + 4e − + O 2 → 2H 2 O · ··(2) [/formula] This reaction contains an oxygen molecule as a reactant in ; thus, the electric current is dependent on the oxygen concentration, assuming that the voltage is constant. This method is widely used to determine renal oxygenation as well as other visceral organ oxygenation, such as the liver. The kidney has a natural oxygen gradient; therefore, data obtained from a microelectrode is dependent on depth. Typically, representative oxygen tensions in the renal cortex and medulla are 50 and 30 mmHg, respectively. This ability to distinguish between the renal cortex and the medulla is an advantage of the microelectrode; however, technical proficiency is required to accurately measure medullary oxygen tension. This technique has two major disadvantages, in addition to its non-applicability in humans. First, this method is highly invasive. The technique requires renal capsule removal and needle insertion into the renal tissue, which results in microbleeding. This method intrinsically hinders repeat measurements within an individual. The second disadvantage is that microelectrodes measure the partial pressure of oxygen from both microcirculation and microbleeding based on the fact that a microelectrode is slightly pulled after insertion to prevent compression. Despite these disadvantages, the use of microelectrodes remains the gold standard for determining tissue oxygen tension because of its superior quantitative performance. To overcome the disadvantages of this established method, novel modifications have been developed. The first involves the use of telemetry [bib_ref] Telemetry-based oxygen sensor for continuous monitoring of kidney oxygenation in conscious rats, Koeners [/bib_ref] [bib_ref] Exogenous and endogenous angiotensin-II decrease renal cortical oxygen tension in conscious rats..., Emans [/bib_ref]. For oxygen tension telemetry, carbon paste electrodes are used to avoid surface poisoning [bib_ref] Characterisation of carbon paste electrodes for real-time amperometric monitoring of brain tissue..., Bolger [/bib_ref]. Once the electrodes are implanted, renal oxygenation can be monitored for 2 weeks without anesthesia. This technique resolves the two major disadvantages inherent to the conventional microelectrode method, including the continuous measurement within an individual and the avoidance of acute effects from general anesthesia. Another approach is urinary oxygen tension measurement . Urinary oxygen tension reflects renal medullary oxygen tension and is useful for monitoring renal oxygenation in critically ill patients [bib_ref] Continuous urine oxygen tension monitoring in patients undergoing cardiac surgery, Kainuma [/bib_ref] [bib_ref] Monitoring renal oxygen supply in criticallyill patients using urinary oxygen tension, Morelli [/bib_ref]. This technique does not require intervention other than the insertion of electrode-equipped bladder catheters; thus, it has the advantage of being easily applied in clinical settings. One disadvantage is the general lack of proof of this in smaller animals. However, a recent study in rabbits reported the ability of urinary oxygen tension measurements to reflect renal medullary oxygen tension [bib_ref] Bladder urine oxygen tension for assessing renal medullary oxygenation in rabbits: experimental..., Sgouralis [/bib_ref]. ## Nitroimidazole probes Pimonidazole is a nitroimidazole. Nitroimidazoles are hypoxic markers that were initially used to detect hypoxic tumors, which are frequently resistant to radiotherapy. Pimonidazole was used to detect tissue hypoxia in a physiological range [bib_ref] Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde..., Arteel [/bib_ref]. Under normoxic conditions, pimonidazole is oxidized by oxygen molecule, whereas pimonidazole is reductively activated under hypoxic conditions. Reduced pimonidazole binds to intracellular thiol-containing proteins. Pimonidazole protein adducts are only detected in hypoxic cells after systemic administration. The detection of pimonidazole protein adducts is typically achieved by immunohistochemistry. Thus, this technique requires the systemic administration of pimonidazole and the preparation of paraffin-embedded sections or frozen sections, which prohibits its use in living animals. Another disadvantage is that this technique reflects the redox state; thus, a redox imbalance independent of the oxygen tension can influence the result. In the kidneys, pimonidazole-positive areas are naturally observed in the medulla and corticomedullar junction [bib_ref] Evidence of tubular hypoxia in the early phase in the remnant kidney..., Manotham [/bib_ref] [bib_ref] Renal cellular hypoxia in adenine-induced chronic kidney disease, Fong [/bib_ref] , which supports the idea that these areas are more hypoxic than the superficial cortex. In CKD models, the pimonidazole-positive area expands [bib_ref] Evidence of tubular hypoxia in the early phase in the remnant kidney..., Manotham [/bib_ref] [bib_ref] Hypoxia and expression of hypoxia-inducible factor in the aging kidney, Tanaka [/bib_ref]. This technique can only detect areas with oxygen tensions lower than a certain threshold (approximately below 10 mmHg); thus, it is neither quantitative nor indicative of the oxygen tension. To overcome the disadvantage of paraffin-embedded sections, several positron emission tomography (PET) tracers with nitroimidazoles have been studied. For example, 18 Ffluoroazomycin arabinoside ( 18 F-FAZA) [bib_ref] Hypoxia-specific tumor imaging with 18F-fluoroazomycin arabinoside, Piert [/bib_ref] was found to accumulate in hypoxic tumors. This probe can be administered in clinical settings, and several papers have proved that it is useful for prognostic prediction in non-small-cell lung cancer [bib_ref] Prognostic value of 18 F-fluoroazomycin arabinoside PET/CT in patients with advanced non-small-cell..., Saga [/bib_ref] [bib_ref] Prognostic significance of hypoxic PET using 18 F-FAZA and 62 Cu-ATSM in..., Kinoshita [/bib_ref]. It remains unclear if this method can successfully detect renal hypoxia in healthy individuals or CKD patients. The difficulty in detecting renal hypoxia is based on the fact that the oxygen tension of non-tumorous organs, including the kidney, is likely to be much milder than that of tumor tissues, even in pathological conditions [bib_ref] PET imaging of cardiac hypoxia: opportunities and challenges, Handley [/bib_ref]. Pimonidazole binds to intracellular thiols at oxygen tensions below 10 mmHg, which is frequently lower than the tensions found in non-tumorous tissues. Another disadvantage is that 18 F-FAZA is physiologically distributed to even normal kidneys and is excreted from the kidney like other PET tracers [bib_ref] Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole, Koh [/bib_ref]. In PET imaging, there is limited resolution with which to distinguish PET probes in the urinary space and intracellular PET probes. However, PET tracing using nitroimidazoles is potentially preferred because this technique is less invasive and suitable for use in CKD patients. Furthermore, this probe reflects the intracellular rather than the extracellular oxygen tension. The optimization of the oxygen tension threshold and biokinetics of PET tracers may overcome the disadvantages listed above and result in new information on renal hypoxia in CKD patients. ## Hif activation detection The cellular response against hypoxia is mainly regulated by HIF; therefore, HIF activation can be used as evidence on tissue hypoxia. One detection method assesses HRE-downstream genes or proteins in tissues. The advantage of this method is that several HIF target proteins are hypoxia markers and affect CKD progression. An example of one of these targets is VEGF. The transcription of the VEGF gene is strongly enhanced under hypoxic conditions. Exogenous VEGF administration ameliorates renal fibrosis in a remnant kidney model, whereas sunitinib administration, a VEGF receptor inhibitor, worsens renal injury in this model [bib_ref] Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor..., Kang [/bib_ref] [bib_ref] Chronic VEGF blockade worsens glomerular injury in the remnant kidney model, Machado [/bib_ref] , although sunitinib also blocks the platelet-derived growth factor receptor. The expression of HIF target genes can be affected by other transcriptional factors and thus is not always affected by the oxygen tension alone. The protein level of VEGF can remain unchanged in some CKD models despite renal hypoxia [bib_ref] Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in..., Futatsugi [/bib_ref]. Classical techniques, such as reverse transcription PCR and immunohistochemistry, require tissue sample preparation. Another method is the in vivo imaging of HIF activity using luciferase. Our group established hypoxia-sensing transgenic rats, in which the luciferase gene was controlled by tandem repeats of HREs located in the 5 ′ UTR of the VEGF gene . A similar approach was employed by Safran et al. who established a hypoxia-sensing mouse strain that expressed firefly luciferase fused to the HIF-1α protein [bib_ref] Mouse model for non-invasive imaging of HIF prolyl hydroxylase activity: assessment of..., Safran [/bib_ref]. These animals emit luminescence after the administration of a systemic chemiluminescent substrate, and the luminescence can be measured from outside the body. This strain was used to detect a decrease in HIF-α hydroxylation induced by hypoxemia and pharmacological PHD inhibition. The advantage of this technique is the lack of autoluminescence in the body, whereas a disadvantage is the reabsorption of luminescence. The kidney contains a significant amount of blood; thus, it can absorb light at an optical wavelength. It is unclear if luminescence from the kidney truly reflects total renal luciferase activity or only superficial activity. have overcome this disadvantage [bib_ref] A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging, Kuchimaru [/bib_ref]. They succeeded in synthesizing a novel luminescent substrate whose wavelength was considerably longer (λ max = 677 nm) than classical luciferins. Owing to its near-infrared wavelength, this substrate can be used to observe deeper tissue and will definitely aid in the intravital imaging of HIF activity. ## Mri-based assessment BOLD-MRI is a useful tool to evaluate renal oxygenation. Here, the spin-spin relaxation rate R2 * is measured, which reflects the deoxyhemoglobin concentration in veins [bib_ref] Non-invasive evaluation of intrarenal oxygenation with BOLD MRI, Prasad [/bib_ref]. To use this technique for tissue oxygenation approximation, we must speculate three proportional relationships: the deoxyhemoglobin concentration and deoxyhemoglobin/total hemoglobin ratio, deoxyhemoglobin/total hemoglobin ratio and partial pressure of oxygen in veins, and partial pressure of oxygen in veins and tissue oxygen level. These three proportional relationships are confounded by the hemoglobin concentration in veins, change in the oxygen affinity of hemoglobin, and intracellular/extracellular oxygen tension dissociation, respectively. Renal anemia and systemic acidbase disorders frequently occur in CKD patients, which may change the oxygen affinity of hemoglobin. The existence of an oxygen shunt in renal arterial and venous vessels suggests that the oxygenation of renal venous vessels is independent of tissue oxygenation. These disadvantages must be taken into account when interpreting data from BOLD-MRI of the kidney. Despite these disadvantages, BOLD-MRI is a promising method because it is a non-invasive technique, particularly when applied in humans. A previous report has shown that the renal R2 * value correlated with eGFR in CKD patients without diabetes [bib_ref] Non-invasive evaluation of kidney hypoxia and fibrosis using magnetic resonance imaging, Inoue [/bib_ref] , whereas another report did not find this correlation in CKD patients [bib_ref] Renal BOLD-MRI does not reflect renal function in chronic kidney disease, Michaely [/bib_ref]. The discrepancy between these two reports may be due to differences in patient backgrounds. Another report has indicated there are several factors other than the oxygen tension, such as blood glucose and uric acid levels, that influence the result of BOLD-MRI, whether directly or indirectly [bib_ref] Determinants of renal tissue oxygenation as measured with BOLD-MRI in chronic kidney..., Pruijm [/bib_ref]. It is debatable whether BOLD-MRI can properly address the heterogeneity of CKD patients. BOLD-MRI, an R2 * -based technique, employs hemoglobin as an oxygen sensor. However, another MRI-based technique that directly measures oxygen molecules has been recently reported [bib_ref] Preliminary study of oxygen-enhanced longitudinal relaxation in MRI: a potential novel biomarker..., O&apos;connor [/bib_ref] [bib_ref] Normal tissue quantitative T1 and T2 * MRI relaxation time responses to..., Winter [/bib_ref]. In this technique, the relaxation rate of the T1 signal (R1) is measured, which is related to the oxygen molecule itself. One report has suggested that R1 in lipids changes with the oxygen tension in mice tumors, whereas another report has shown that the R1-based technique is less sensitive in detecting renal hypoxia in hypoxemia than the R2 * -based technique [bib_ref] Mapping of oxygen by imaging lipids relaxation enhancement: a potential sensitive endogenous..., Jordan [/bib_ref] [bib_ref] T2 * and T1 assessment of abdominal tissue response to graded hypoxia..., Ganesh [/bib_ref]. Together with the low invasiveness of MRI-based techniques and the limitation of BOLD-MRI, R1-based measurement is a potentially viable method because it directly determines the oxygen tension. ## Phosphorescence Phosphorescence is a type of light emitted from a molecule in the triplet excited state. Molecules in this state can lose their energy by being hit by oxygen molecules. Phosphorescence intensity and phosphorescence lifetime depend on the oxygen concentration [bib_ref] Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused..., Rumsey [/bib_ref]. Thus, this technique can directly measure the oxygen tension. Substances that emit phosphorescence are limited, and exogenous phosphorescence dyes are used to measure phosphorescence. Notably, the phosphorescence lifetime is far longer than the fluorescence lifetime. Both phosphorescence intensity and phosphorescence lifetime can be quantitatively measured. However, phosphorescence dye concentrations in the target organ are required to determine the oxygen tension in vivo using the phosphorescence intensity. Therefore, phosphorescence lifetime measurements are frequently used to determine the oxygen tension in vivo [bib_ref] Monitoring of renal venous PO 2 and kidney oxygen consumption in rats..., Mik [/bib_ref] [bib_ref] Imaging local neuronal activity by monitoring PO 2 transients in capillaries, Parpaleix [/bib_ref] [bib_ref] Direct measurement of local oxygen concentration in the bone marrow of live..., Spencer [/bib_ref] [bib_ref] Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement, Hirakawa [/bib_ref]. The phosphorescence lifetime does not depend on dye concentration as long as it sufficiently accumulates. In phosphorescence lifetime measurements, the Stern-Volmer equation is used: [formula] [O 2 ] = 1 k q 1 τ − 1 τ 0 · ·· (3) [/formula] where k q is the rate constant, τ is the phosphorescence lifetime at the oxygen concentration of [O 2 ], and τ 0 is the phosphorescence lifetime in an anoxic condition [bib_ref] An optical method for measurement of dioxygen concentration based upon quenching of..., Vanderkooi [/bib_ref]. These two constants, k q and τ 0 , depend on the phosphorescence dye and circumstantial microcondition, such as solvent. Thus, the determination of these parameters is problematic. The oxygen dependence of the phosphorescence lifetime was established in the 1980s. It is utilized with fluorescence needle probes, which contain an oxygen-sensitive fluorescence dye, such as protoporphyrin IX (PpIX) described below, which has similar characteristics against oxygen. Several studies have employed this fluorescence needle probe because of its high sensitivity to the oxygen tension [bib_ref] Renal medullary tissue oxygenation is dependent on both cortical and medullary blood..., O&apos;connor [/bib_ref] [bib_ref] Early changes with diabetes in renal medullary hemodynamics as evaluated by fiberoptic..., Santos [/bib_ref]. However, this method cannot overcome the two major disadvantages of microelectrodes: unclear measurement site and high invasiveness. Thus, another way to apply phosphorescence lifetime measurement to in vivo O 2 measurement is warranted, and currently, the administration of phosphorescence dyes that are distributed in a certain tissue or cell is frequently used. One research milestone was the report by [bib_ref] Direct measurement of local oxygen concentration in the bone marrow of live..., Spencer [/bib_ref]. They employed a water-soluble phosphorescence dye, PtP-C343, to measure the oxygen tension in arterioles penetrating cortical bones and showed that the oxygen tension decreased when the arteriole entered the medullary canal in mice. This research directly proved the lower oxygen tension in the bone marrow, which was hypothesized based on the unique vasculature system in the marrow and pimonidazole protein adduct immunohistochemistry [bib_ref] Metabolic regulation of hematopoietic stem cells in the hypoxic niche, Suda [/bib_ref] [bib_ref] Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status..., Nombela-Arrieta [/bib_ref]. Powerful research on the kidney was published by [bib_ref] Monitoring of renal venous PO 2 and kidney oxygen consumption in rats..., Mik [/bib_ref]. They used Oxyphor G2 as a water-soluble phosphorescence dye and proved that oxygen tension in renal vein was decreased after endotoxin shock in rats. This work clarified where the oxygen tension should be measured (i.e., in renal veins). The phosphorescence dye is excited only in blood in renal vein. In this study, there is no phosphorescence signal from arterial blood, capillary blood, or interstitial fluid, which is problematic in the study of solid organs. The aforementioned phosphorescence dyes are distributed in the extracellular fluid, mainly in the blood; thus, these dyes cannot directly determine the intracellular oxygen tension. Our group recently reported a novel technique for measuring the intracellular oxygen tension using BTPDM1, a cationic lipophilic phosphorescence dye . In the kidney, this dye is found only inside tubular cells. Thus, phosphorescence lifetime imaging reflects only the intracellular oxygen tension in tubules. Using this method, phosphorescence lifetime measurement revealed renal hypoxia in CKD, and the phosphorescence lifetime was successfully converted to the partial pressure of oxygen using the calibration curve obtained from a cultured tubular cell line. One disadvantage of this technique is dye toxicity when applied in human beings because BTPDM1 contains iridium, a heavy metal. Another dye that may overcome this disadvantage is PpIX [bib_ref] Mitochondrial PO 2 measured by delayed fluorescence of endogenous protoporphyrin IX, Mik [/bib_ref]. PpIX is a precursor of heme and is unique in that it can emit delayed fluorescence. This delayed fluorescence is similar to phosphorescence in terms of oxygen sensitivity, i.e., its phosphorescence lifetime depends on the oxygen tension. PpIX localizes to the mitochondria, and its physiological concentration is so low that it cannot be used as an endogenous oxygen sensor. However, when a large amount of 5-aminolevulinic acid (5-ALA), a PpIX precursor, is administered, the cellular synthesis of PpIX sufficiently increases to allow its use as an oxygen sensor via delayed fluorescence lifetime measurements. Using this method, the topical application of 5-ALA enables PpIX accumulation in the skin of healthy human volunteers and the oxygen tension estimate in human skin [bib_ref] Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial..., Harms [/bib_ref]. Thus, this technique can be applied to abdominal organs, such as the liver [bib_ref] Microvascular and mitochondrial PO 2 simultaneously measured by oxygen-dependent delayed luminescence, Bodmer [/bib_ref]. These phosphorescence or delayed fluorescence lifetime measurement techniques have the advantage of direct oxygen measurements and a superior quantitative performance. In contrast, these techniques cannot be used for assessing the deeper portion of the kidney, such as the outer medullary region, because phosphorescence is absorbed in tissues, particularly by hemoglobin. ## Conclusion and future perspectives In this review, we addressed the pathophysiological importance of renal hypoxia with a focus on hypoxia detection. Although oxygen tension determination has been studied for a long time, recent advances have made it possible to achieve continuous measurements, non-invasive oxygen assessments, and intracellular oxygen assessments. All these methods have their advantages and disadvantages; therefore, a comprehensive understanding and proper selection of the available methods are required to assess renal hypoxia and identify the final common pathway in individual hypoxia patients. Thus, a sufficient understanding of oxygen detection techniques will help researchers develop new drugs against CKD and renal hypoxia. # Author contributions YH wrote the manuscript and TT and MN edited the manuscript. [table] TABLE 1 |: Comparison of hypoxia detection methods. [/table]

Keratoconus with Central Serous Chorioretinopathy: A Rare Combination Keratoconus and central serous chorioretinopathy are two rare diseases. They can occur together in some individuals. We report a case of a 48-year-old man, who presented to our clinic with decreased visual acuity on his left eye. Physical examination, biomicroscopy, corneal topography, and optical coherence tomography revealed keratoconus and central serous chorioretinopathy. We discuss the possible connection between these two conditions. # Introduction Keratoconus causes visual loss due to increasing irregular corneal astigmatism and affects 86 in 100,000 people. It is an idiopathic, noninflammatory, progressive corneal ectasia characterized by bilateral, usually asymmetric thinning of the cornea. The onset typically is in teenage years, and it is much less common in middle-aged and elderly individuals. While it often presents as an isolated condition, keratoconus may also be associated with many systemic disorders and/or ocular diseases. Central serous chorioretinopathy (CSC) is a major cause of vision threat among middle-aged male individuals. It is a secondary idiopathic serous neurosensory detachment, believed to be due to a leakage of fluid from the choroid through tight junctions between adjacent retinal pigment epithelial cells (RPE). Its precise pathogenesis is unknown, but there are some possible risk factors relating to the occurrence of CSC, such as H. pylori infection, steroid use, sleep disorders, autoimmune disease, psychotropic medication use, and type-A behavior. To the best of our knowledge, no association between CSC and corneal diseases has been described. Here, we present the case of a patient with keratoconus and central serious chorioretinopathy (CSC). ## Case report 2.1. History. A 48-year-old male was referred to our ophthalmology clinic with decreased visual acuity on the left eye. His medical history included Crohn's disease since 2005, seasonal allergic rhinoconjunctivitis, and prostate cancer status post prostatectomy in 2016. He had been treated with methylprednisolone intermittently therapy for the last 4 years. Five years ago, he had an episode of episcleritis treated with dexamethasone (1%) drops. He was very anxious. His family history was unremarkable. ## Measurements 2.2.1. Entrance Tests, Refraction, and Binocularity. Best corrected distance visual acuity was 10/10 in the right eye and 2/10 in the left eye (Snellen chart). Ocular motility was normal. Visual fields were intact to confrontation visual field exam (Donders' test). His pupils were equal, round, and reactive to light, with no afferent pupillary defect. Color vision discrimination, using the Ishihara color vision test, showed normal color vision in each eye. The red-cap color comparison was equal between the two eyes. Automated keratometry revealed of -1.00 sph and -1.50 cyl at 90°in the right eye and -5.00 cyl at 120°in the left eye. 2.2.2. Biomicroscopy. The eyelids, eyelashes, conjunctiva, iris, and anterior chamber appeared normal. Anterior segment biomicroscopy showed mild corneal thinning inferior to the pupil in the left eye. There were no other signs of keratoconus (Fleischer's ring, Vogt's striae, Rizzuti's sign, oil drop). There was no stromal edema or corneal opacities in either eye. He had incipient posterior subcapsular cataract in both eyes. Fundus examination of both eyes was normal. Vitreous, optic nerves, vasculature, and peripheral retina in both eyes were unremarkable and normal for his age. The intraocular pressure (as measured by Goldmann applanation tonometry) was 30 mmHg in the right eye and 28 mmHg in the left eye. ## Corneal topography and optical coherence tomography (OCT). The anterior surface corneal topography of the left eye showed normal K1 and K2 values, but the difference between Kmax-K2 was >1D. For the right eye, the simulated minimum keratometry reading had normal K1 and K2 values and the difference between Kmax-K2 was abnormal as the left eye. Secondary, Kmax (do)-Kmax (so) was >2D. Corneal pachymetry measurements at the thinnest locations were 477 microns and 504 microns for the left and right eye, respectively . The OCT examination demonstrated fluid accumulation between the interdigitation zone (IZ) and the retinal pigment epithelium (RPE) as well as increased choroidal thickness. The junction between photoreceptor inner and outer segments (IS/OS) was not detected in the detached neurosensory retina. The outer photoreceptor layer of the detached neurosensory retina above the clear subretinal space was irregularly thickened and granulated . The retinal thickness ILM was increased in the horizontal meridian . Clinical findings were characteristic of keratoconus, central serious chorioretinopathy, and elevated intraocular pressure, as a result of steroid therapy. ## Treatment. Aldosterone antagonists are effective in decreasing subretinal fluid and improving visual acuity in patients with CSC 9 . We prescribed the selective aldosterone antagonist eplerenone which is associated with a favorable side effect profile. Beta blockers are not the most potent medication of IOP lowering, but they are efficacious, well tolerated by most patients and a low cost-effective choice, making them a good choice as a first-line agent in case of the absence of any contraindications. They induce b receptor blockage which lowers aqueous humor formation and secretion especially during daytime. Timolol is one of the most popular and prescribed antiglaucoma beta blocker agents, as a first-line drug in most forms of open angle glaucoma and ocular hypertension. Our patient had elevated IOP due to chronic use of systemic steroids as part of treat-ment for his Crohn's disease. Given the necessity for ongoing steroid therapy, we elected to prescribe timolol to lower IOP (probably steroid responder). Spectacles were, also, prescribed for keratoconus to improve his vision. # Discussion This case report demonstrates the coexistence of keratoconus and central serous chorioretinopathy to the left eye of a 48year-old Caucasian male with Crohn's disease. Keratoconus and CSC are diseases that affect different parts of the eye. Keratoconus may be associated with many systemic disorders such as diabetes, asthma, Down syndrome, collagen vascular disease, and sleep apnea. There are some strong associations between keratoconus and several autoimmune diseases and allergic disorders. Furthermore, patients with inflammatory bowel diseases may have an increased risk of keratoconus. The main treatment of inflammatory bowel disease is corticosteroids. They are administered topically, orally, or intravenously and rapidly and consistently improve moderate to severe active ulcerative colitis and Crohn's disease. There is no known association between corticosteroids and keratoconus. In vitro trials of dexamethasone on human cornea showed that dexamethasone significantly increases keratocyte proliferation, but it also induces apoptosis of cultured keratocytes at any concentration. Keratocyte apoptosis is associated with keratoconus, in which keratocyte density is lower compared to healthy controls. On the other hand, central serous chorioretinopathy has been associated with most routes of steroid administration. The majority of the literature on CSC and steroid medication suggests a significant association. At the current time, the pathophysiology of CSC is poorly understood. To our knowledge, there is no direct association between these two conditions. The subfoveal choroid is diffusely thickened in patients with CSC likely because of the choroidal vascular dilatation. In addition, the subfoveal choroid in keratoconus eyes is thicker than that in healthy population. A genetic disorder could better explain the coexistence of keratoconus and central serous chorioretinopathy, especially in predisposed individuals. Keratoconus is associated with extensive alteration in the expression of genes coding collagen type IV, fibronectin, laminin, lysyl oxidase, and tissue inhibitor of metalloproteinase 3 (TIMP-3) involved in cellmatrix, cell-cell interactions, massive changes of the cytoskeleton, extracellular matrix remodeling, and transmembrane signaling. The TIMPs are multifunctional proteins, with different cellular effects which are not well understood. TIMP-3 is the only TIMP protein that binds tightly to the extracellular matrix. Immunohistochemistry showed TIMP3 immunostaining was dense, specifically localized in Bruch's membrane and some choroidal vascular basement membranes. Increased expression of TIMP3 could contribute to the activation of apoptotic cell death processes through restructuring of the architecture, and disruption of photoreceptor-matrix interactions. Altered expression of TIMP-3 Case Reports in Ophthalmological Medicine could modify the RPE basement membrane, causing variation in the pigment epithelial integrity. These observations could explain the disturbance of the outer blood-retinal barrier. Reduced expression of some proteins to RPE due to genetic disorders could explain a relationship between the two diseases, potentially reflecting a dysfunction of epithelial layers and basement membranes. More studies are necessary to elucidate a possible connection between these diseases. # Conclusion According to the literature, there is no known association between keratoconus and CSC. Genetic predisposition seems to be a plausible mechanism to explain the occurrence of both conditions on the same individual. More studies are necessary to elucidate the possible connection between these diseases. ## Abbreviations CSC: Central serous chorioretinopathy IZ: Interdigitation zone IOP: Intraocular pressure IS/OS: Inner and outer OCT: Optical coherence tomography RPE: Retinal pigment epithelium. ## Consent The patient consented to the publication of the case in writing. # Disclosure All authors attest that they meet the current ICMJE criteria for authorship. ## Conflicts of interest None of the authors have financial disclosures.

Barriers to and Facilitators of Human Papillomavirus Vaccination Among People Aged 9 to 26 Years: A Systematic Review Background: Cervical and oropharyngeal cancers are associated with human papillomavirus (HPV) infection, which can be prevented with the vaccines. However, uptake of the HPV vaccine remains low in many countries. There is a need to better understand the barriers to and facilitators of HPV vaccination from young people's perspectives.Methods: Five electronic databases were searched for original publications (dated January, 2006-December, 2019) reporting barriers to and facilitators of HPV vaccination among young people. All articles were screened against prespecified eligibility criteria, and data were extracted against prespecified form.Results: A total of 13 studies that were published in international peer-reviewed journals and met the stated eligibility criteria were identified. The barriers reported were centralized around lack of knowledge about HPV and the HPV vaccine, fear about the safety and efficacy of the HPV vaccine, fear about not being able to pay for the HPV vaccine, and discrimination regarding to the HPV vaccine. The facilitators reported were centralized around trust in the efficacy and safety of the HPV vaccine, discounted price of vaccination, positive recommendations from others, perceived risk of HPV infection, and benefits of vaccine.Conclusions: After their introduction 14 years ago, knowledge deficiency of the HPV vaccine is still a critical barrier to vaccination. Educational initiatives aimed at adolescents and young adults were urgently needed. Understanding factors that arbitrate in early HPV vaccination is critical for improving the HPV vaccination rate. P ersistent infection with high-risk types of human papillomavirus (HPV) is known to cause cervical, oropharyngeal, vaginal, vulvar, penile, anal, and rectal cancers.Among them, cervical cancer has the highest incidence (7.4 per 100,000), and the second is oropharyngeal cancer (4.5 per 100,000).Approximately 99.7% of cervical cancers and 80% of oropharyngeal cancers are now attributed to high-risk types of HPV.Two international studies show the decreasing incidence rate of cervical cancer and the rising incidence rate of oropharyngeal cancer.It is noteworthy that cervical cancer incidence remains high, although a declining trend was observed.Fortunately, cervical and oropharyngeal cancers are vaccine-preventable diseases.Since 2006, many pivotal clinical trials have shown that both bivalent and quadrivalent HPV vaccines have significantly high effectiveness (>90%) in preventing HPV infections and related diseases caused by vaccine-targeted HPV genotypes.The Advisory Committee on Immunization Practices recommends HPV vaccine initiation at ages 9 to 26 years.One study on modeled evaluations of the cost-effectiveness of HPV vaccination indicated that an HPV vaccination coverage of 70% in women has been regarded as the threshold for optimum cost-effectiveness.As of October 2019, 100 countries and territories have introduced the HPV vaccine into their national immunization schedules.However, global estimates of HPV vaccination coverage indicated that only 33.6% of young females aged 10 to 20 years had received the full course of the HPV vaccine in more developed regions compared with only 2.7% of females in less developed regions.There is a huge gap between real-world HPV vaccine coverage and recommended threshold coverage. Identifying barriers and facilitators to HPV vaccination from key stakeholders' perspectives is paramount. Barriers at the policy level mainly include nonmandatory HPV vaccination and incomplete insurance coverage.Health care providers and parents both reported knowledge gaps and financial concerns as barriers.The former also mentioned parents' negative attitudes regarding vaccination. In contrast, parents reported not receiving caregivers' recommendations and concerns about vaccine safety as their additional barriers to HPV vaccination.There are many studies about providers' and parents' perceived barriers to having their patients and children vaccinated, but few studies focus on the vaccine recipient's own perspectives. To increase the uptake of HPV vaccination, a better understanding of the factors that obstruct early HPV vaccination, as experienced by the vaccine recipient, is urgently needed. The purpose of this study is to systematically review self-reported barriers and facilitators to HPV vaccination among young men and women aged 9 to 26 years to inform future efforts to improve HPV vaccination initiation and uptake. The data from this study will enable more precise and accurate assessments of vaccination experience from the vaccine recipient perspective so that future interventions can be developed to improve the HPV vaccination rate and cancer prevention. # Methods A protocol for this systematic review was not registered with a database. However, this review was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (Supplemental File 3, http://links.lww. com/OLQ/A648). ## Search strategy We conducted a systematic review of the literature to summarize the barriers to and facilitators of HPV vaccination among adolescents and young adults. Given the HPV vaccine became available in 2006, we included only peer-reviewed journal articles published from January 1, 2006, to December 31, 2019. Five databases, namely, PubMed, Web of Science, Scopus, Medline (EBSCOhost), and PsycINFO (EBSCOhost), were searched for studies published in English. All searches were conducted in a combination of Medical Subject Headings and free terms including "papillomavirus vaccines," "vaccination," "cross-sectional studies," "young adult" and "adolescent." A medical librarian was consulted to verify the search strategy, and the search was customized for each database separately. See Supplementary File 1 (http://links.lww.com/OLQ/A646) for the complete retrieval strategy. ## Eligibility screening To be eligible for inclusion, studies had to report perceived barriers and facilitators of HPV vaccination among young people aged 9 to 26 years. Studies that described the HPV vaccine uptake, and attitudes toward or knowledge about the HPV vaccine without reporting the barriers and facilitators to HPV vaccination were excluded. Studies that included solely parents, health care providers, and state leaders, without enrolling adolescents and young adults aged 9 to 26 years were also excluded. In addition, our review also excluded sexual minority populations including transgenders or gay or lesbian individuals because their barriers and facilitators to HPV vaccination should be substantially different from the general adolescents and young adults. Primary studies that used qualitative, quantitative, and mixed methods were included in the review. Review articles, case reports, abstracts, and conference proceedings were excluded. ## Data extraction The search was performed independently by 2 authors. The authors screened the titles and abstracts of articles retrieved in the initial search based on the inclusion criteria of this study. Full texts of relevant studies were then screened for the final inclusion. Disagreements between the authors were resolved by consensus. To gather all existing evidence, we extracted data into a prespecified data extraction formed by L.Z. and checked by J.W., including country, objectives, study design, respondents, and sample size. We also extracted the self-reported barriers to and facilitators of HPV vaccination mentioned by young respondents. ## Quality assessment The quality and validity of each article comprising this analysis were assessed using the Agency for Healthcare Research and Quality (AHRQ) criteria for observational studies, which contains 11 items.All questions were answered "yes" (scored as 1), "no" (0), or "unclear" (0). Rating criteria for the AHRQ were as follows: low quality, 0-3; moderate quality, 4-7; and high quality, 8-11. # Data analysis Data were collected from 13 included studies using Microsoft Excel (Microsoft), with respect to study characteristics, quality assessment, and study results. Given the heterogeneity of included studies, we conducted a narrative synthesis of abstracted data instead of statistical meta-analysis. # Results ## Characteristics of included studies Using the predefined search terms, 568 potential articles were identified. After initial review for relevance and duplication, 440 abstracts remained to be screened for eligibility. A total of 151 articles were retrieved for full-text analysis using the same inclusion criteria. The characteristics of all included studies are shown in. Of the 13 articles, 2 were conducted by qualitative interviews, and the remainder were questionnaire-based surveys in study design. All studies were published in English and conducted after licensure of the HPV vaccine (after 2006). The mean age of survey respondents in 13 included articles was younger than 26 years. The geographical distribution of these studies was as follows: the United States (N = 2), Malaysia (n = 2), Hong Kong, China (n = 1), United Arab Emirates (n = 1), Germany (n = 1), Uganda (n = 1), India (n = 1), Greece (n = 1), Canada (n = 1), Brasilia (n = 1), and Singapore (n = 1). Five included studies only examined females, 2 studies only examined males, and the other studies included both males and females. All of the studies provided information on barriers, whereas only 4 studies reported facilitators in their decision-making process. As shown in, the analysis using the AHRQ criteria demonstrated that the studies were of good quality, as the eligible articles were all of moderate or high quality. All 13 articles scored between 4 and 8 points on the quality measure. ## Perceived barriers from adolescents and young adults At this level, we organized the study results into the following primary categories: (1) lack of knowledge about HPV and the HPV vaccine, (2) fear about the safety and efficacy of the HPV vaccine, (3) fear about not being able to pay for the HPV vaccine, (4) discrimination regarding to the HPV vaccine, and (5) other barriers, such as lack of time, fear of pain, and a negative experience with vaccinations in general . Nearly all included articles (11/13) reported that a lack of knowledge on HPV, cervical cancer, and HPV vaccines affected adolescents' willingness to initiate HPV vaccination programs. According to our findings, HPV-related knowledge among people aged 9 to 26 years is rather limited. In 2012, 6 years after the first HPV vaccine officially launched by the Food and Drug Administration, there were still people who said they never heard about HPV vaccination.Some people said they were not aware of the HPV vaccine and felt shy about discussing it with parents or health care providers.People may also be misled by some messages, such as that students (aged 9-26 years) are too young for vaccination.We also found that many people think vaccination is not deemed necessary because of the lack of a perceived risk of HPV infection.They said that they were not sexually active and were not prone to infection.Moreover, there was little difference between medical and nonmedical students in accessing HPV-related information. Many studies (10/13) on factors influencing the intention of adolescents or young adults to get HPV vaccines reported concerns about the safety and efficacy of the HPV vaccine. Concerns about adverse effects were the most common reason for rejecting HPV vaccinations among people aged 9 to 26 years. More than half of the included articles (8/13) reported concern about adverse effects as a barrier to receiving HPV vaccination. Students from Malaysia indicated that they also had fears about the safety of vaccine and thought it was still new.One study on undergraduate women indicated that they had not yet been vaccinated because they do not know enough about the vaccine's potential adverse effects and whether the vaccine works. 31s Even medical students also reported doubts about the efficacy of the HPV vaccine.More than two-thirds of studies (9/13) reported that the cost of vaccines was a concern. 21,23,26-30,31s,32s Studies conducted in developed regions (the United States, Greece, Canada, Singapore, and Hong Kong, China) refer to financial issues as a barrier to receiving the HPV vaccine; this was also a concern in some developing countries (Malaysia, India, Brasilia). Among these, 2 questionnaire surveys conducted in America and Canada both noted that the vaccine costs too much and is not covered by personal health insurance. 31s,32s Moreover, one study conducted in Malaysia also reported that HPV vaccination was not cost-effective.Nearly half of the articles (6/13) reported that discrimination against the HPV vaccine also influenced the decision regarding vaccination. Previous research found that others' recommendations against vaccination played a key role in the decision regarding HPV vaccination. Some students said that their parents would not allow them to take the vaccine; notably, vaccination against HPV is not recommended by some physicians either. Negative perceptions about HPV will spread throughout the community. 33s In addition, discrimination regarding sexually transmitted diseases from religious authority was also reported as a barrier to HPV vaccination.One study conducted in Greece explicitly described HPV vaccination as a religious taboo.In addition to the 4 main barriers mentioned previously, there are also some other obstacles that will be described in detail here. First, questionnaire surveys on young women reported that fear of pain was one of the reasons for refusing HPV vaccination. Moreover, most people aged 9 to 26 years were still attending school, whose busy schedule would preclude them from talking to health care providers about vaccination or complete all 3 doses of the HPV vaccine. Finally, some people also mentioned medical contraindications and not knowing where to get vaccinated as their reasons for not initiating HPV vaccination. ## Perceived facilitators from adolescents and young adults At this level, we organized the study results into the following primary categories: (1) trust in the safety and efficacy of the HPV vaccine, (2) discounted price of vaccination, (3) positive recommendations from others, and (4) perceived risk of HPV infection and benefits of vaccine . Many students reported that recommendations from others have a positive impact on increasing vaccination uptake and credible sources, including doctors, parents, friends, and religious authority.Otherwise, awareness of vaccines' preventive role against cervical cancer, for self and others, was a facilitator of receiving the HPV vaccine. # Discussion Although there is substantial literature on factors associated with higher and lower HPV vaccination uptake, the body of literature reporting barriers from the vaccine recipient's perspectives that are critical to HPV vaccination is relatively sparse. This is a systematic review that reviews the literature on barriers to and facilitators of HPV vaccination among adolescents and young adults. Our systematic review found that the barriers to vaccination were mainly concerns about the lack of knowledge about HPVand the HPV vaccine, fear about the safety and efficacy of the vaccine, fear about not being able to pay for the HPV vaccine, and discrimination regarding the HPV vaccine. Second, trust in the efficacy and safety of the HPV vaccine, discounted price of vaccination, positive recommendations from others, perceived risk of HPV infection, and benefits of the vaccine are the facilitators of HPV vaccination. The lack of knowledge or information on cervical cancer and HPV infection was the most commonly reported barriers when considering the uptake of HPV vaccination. This provides an opportunity for the spread of misinformation in social networks. Compared with other populations, adolescents and young adults are more vulnerable to misinformation. For instance, many adolescents refused to receive the HPV vaccine because they indicated that they were not sexually active and were not prone to infection. Unfortunately, approximately 80% of women will acquire HPV infection during their lifetime. 34s As a result, educational initiatives aimed at people aged 9 to 26 years may be most successful if designed to increase awareness of susceptibility to HPV infection and HPV transmission. School-based meetings are perhaps an essential sensitization strategy to increase the amount and quality of knowledge and information on cervical cancer and the HPV vaccine. 35s Previous studies have shown that integrating school immunization provision with general practice provides a convenient location for parents and their children to discuss the immunization program. Our results also indicated that concerns about the safety and efficacy of the HPV vaccine constitute a barrier to HPV vaccination among the target population. In contrast, trust in the efficacy and safety of vaccines may play a role as vaccination activators. In fact, many randomized controlled trials have confirmed the safety and efficacy of HPV vaccination. 37s-39s No serious vaccine-related adverse effects were reported in the clinical study. 37s As previously reported, concerns about the safety and effectiveness of the vaccine is related to lack of knowledge. 40s This knowledge gap reveals an additional priority for education. Health literacy initiatives in adolescents and young adults should focus on the efficacy of the HPV vaccine and highlight its established safety at the same time. Another barrier is the cost of vaccination. In contrast, the discounted price of vaccination has a positive effect on initiating [formula] Y N Y Y N Y Y Y N Y N 7 Ortashi et al. 22 Y N Y Y N Y Y N N Y N 6 Lee et al. 23 Y Y Y Y N Y Y Y N Y N 8 Remschmidt et al. 24 Y N Y Y N Y Y Y N Y N 7 Turiho et al. 25 Y Y Y N N Y Y Y N Y N 7 Swarnapriya et al. 26 Y N Y Y N Y Y Y N Y N 7 Mammas et al. 27 Y Y Y Y N Y Y N N Y N 7 Fernandes et al. 31s Y N Y Y N Y Y Y Y Y N 8 Wanderley et al. 28 Y N Y Y N Y Y N N Y N 6 Widjaja 29 Y N N Y N Y Y N N Y N 5 Schmidt-Grimminger et al. 33s Y N N N N Y Y N N Y N 4 Zhuang et al. 30 Y N N Y N Y Y N N N N 4 Katz et al. 32s Y N N Y N Y Y N N N N 4 [/formula] Criteria: yes (Y), 1; no (N), 0; unclear (U), 0. 1 = Define the source of information (survey, record review); 2 = list inclusion and exclusion criteria for exposed and unexposed subjects (cases and controls) or refer to previous publications; 3 = indicate time period used for identifying patients; 4 = indicate whether or not subjects were consecutive if not population-based; 5 = indicate if evaluators of subjective components of study were masked to other aspects of the status of the participants; 6 = describe any assessments undertaken for quality assurance purposes (e.g., test/retest of primary outcome measurements); 7 = explain any patient exclusions from analysis; 8 = describe how confounding was assessed and/or controlled; 9 = if applicable, explain how missing data were handled in the analysis; 10 = summarize patient response rates and completeness of data collection; 11 = clarify what follow-up, if any, was expected and the percentage of patients for which incomplete data or follow-up was obtained. HPV vaccination. Previous studies have also shown that the highest HPV vaccination coverage rates are observed in countries where vaccines are funded from the national budget. 41s This was the case in Japan, where a high uptake rate for individual HPV vaccination was obtained. The main source of HPV vaccination program costs in developed countries is the government budget, whereas in developing countries, international donors such as the Global Alliance for Vaccines and Immunization, can be an important source. 43s Actually, a record low price of as little as US $4.50 per dose for low-income countries compared with more than $100 in high-income countries was announced by the Global Alliance for Vaccines and Immunization as early as 2013. Additional obstacles to vaccination include discrimination against the HPV vaccine and practical barriers. Discrimination against the HPV vaccine refers to religious taboo, cultural biases; practical barriers refer to fear of needles, busy schedule, limited access to health services, medical contraindications. Many of these factors are related to all kinds of vaccines. However, cultural and religious sensitivity seems to be specific to the HPV vaccine. Two studies conducted in the United Arab Emirates and Singapore mentioned religious authority as a barrier to HPV vaccination.A previous study has also revealed that sex-related issues are taboo topics in many countries around the world, especially in Asian communities. 45s However, a survey conducted in Scotland indicated that some women would like to receive the information on HPV delivered within the religious community, not within other places for social gathering. 46s Therefore, health literacy initiatives addressing HPV vaccination gaps also needs to be culturally tailored. It is perhaps a good approach that develop education programs with the help of local community and religious leaders. Of the 4 facilitators of vaccination mentioned in our review, positive recommendations from others were most commonly reported. Adolescents and young adults usually receive recommendations for HPV vaccination from their physicians, parents, and friends. Furthermore, health care providers' recommendations are more convincing to the vaccine recipient than recommendations from other sources. 47s According to a study of 17,264 girls aged 12 to 17 years in the United States, girls who receive a clinician recommendation to vaccinate are 23 times more likely to be vaccinated than those not counseled. 48s It is also notable that physician effects could be both positive and negative. Thus, acceptability for vaccination of adolescents and young adults could be improved by increasing providers positive recommendations. Physicians should use this influence to disseminate HPV-related knowledge to youths, including the transmission of virus, the likelihood of infection, and its oncogenicity. Our findings should be interpreted with the following limitations. First, we only included peer-reviewed studies published in the English language. Therefore, gray literature was excluded from the review, which may have biased the results. Second, all studies were cross-sectional, precluding an understanding of changes in knowledge, attitudes, and practices over time. In addition, most studies recruited convenience samples, thus limiting the generalizability of the findings. Furthermore, another limitation relates to the medical background of the study population. A quarter of the retrieved articles concern studies performed in medical or pharmacy student populations. However, nearly three-quarters of the studies were conducted in populations without a medical background. This knowledge gap may also affect outcome. This review provides valuable data about adolescents' and young adults' self-reported barriers and facilitators to initiating HPV vaccination. After their introduction 14 years ago, knowledge deficiency of the HPV vaccine is still a critical barrier to vaccination. Educational initiatives aimed at adolescents and young adults were urgently needed. School-based meetings and health care

Case report Introduction: Amyloid light chain (AL) results from the deposition of immunoglobulin light chain fragments, and can affect multiple organs/systems. Our patient was diagnosed as scleroderma repeatedly because of extensive skin thickening and hardening, but the treatment was not effective. We did extensive laboratory examinations including serum/urine protein electrophoresis and flow cytometry assay of bone marrow aspiration.Conclusion: A diagnosis of primary AL amyloidosis was established.Abbreviations: AL = amyloid light chain, MG = monoclonal gammopathy, MGUS = monoclonal gammopathy of undetermined significance, MM = multiple myeloma, MRA = magnetic resonance angiography, MRI = magnetic resonance imaging. # Introduction The amyloidosis is a group of rare diseases caused by extracellular deposition of amyloid. [bib_ref] Systemic amyloidosis, Wechalekar [/bib_ref] It may affect multiple organs with protean manifestations, thus often causing delayed or incorrect diagnoses. While nephrotic syndrome, cardiomyopathy, and peripheral neuropathy are common in amyloidosis, it is relatively rare for a patient with the major presentations as myopathy and extensive skin involvement. Here, we report a case of a middle-aged woman who was hospitalized because of the difficulties in squatting and tongue movements, and sclerodermalike changes. This report had been approved by the Ethics committee of the First Affiliated Hospital of Zhengzhou University. ## Case report A 50-year-old female patient without the history of any diseases was admitted to our hospital in August 2016. She had difficulties in squatting for 5 years, impaired tongue movements, coarseness and dysphagia for 8 months, extensively tightened and pigmented skin for 6 months. She felt tingling when touching cold water and warming up hands relieved the symptom. The difficulty in squatting was noticed in year 2011 and the symptom kept progressing to the point where the skin became so extensively tightened that the movements of lower limbs were significantly restricted. Constitutional symptoms included low fever, fatigue, joint pain, and morning stiffness. No Raynaud phenomenon and joint swelling were noticed. She once visited a physician at local hospital where she was diagnosed as scleroderma. She was treated with traditional Chinese medicines and very little improvement was achieved. Two years ago, she had snoring and apnea during sleep and was diagnosed as obstructive sleep apnea hypopnea syndrome, but she refused noninvasive ventilation. Eight months ago, she visited a neurologist in other hospital due to difficulty in opening mouth and moving tongue, dysphagia, hoarseness, and ever-worsening skin tightness. Brain magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) showed bilateral lacunar infarction of basal ganglia, white matter demyelination in mild cerebral, multiple sclerosis in cerebral arteries, and mild stenosis of bilateral posterior cerebral artery. Neuro-electrography revealed mild to moderate demyelinating peripheral neuropathy and the damage in the left pyramidal tract. Polysomnography revealed a moderate obstructive sleep apnea-hypopnea syndrome and mild hypoxemia. She was then diagnosed as extrapyramidal disease and sleep apnea-hypopnea syndrome, and treated with madopar tablet and neurotropin. However, no evident effect was observed. Six months ago, she went to Beijing Union Hospital, the best hospital in China, because of flake skin pigmentation on her face, upper limbs, low back, and buttock areas. She was diagnosed as systemic sclerosis and treated with prednisone, Tripterygium glycosides, and methotrexate for 2 weeks. No improvement was achieved. The patient was admitted to our hospital on August 24, 2016. Routine physicals showed normal vital signs. Extensive skin pigmentation was found in the above-mentioned areas. The skin became that so thickened and tightened that limb movements were seriously limited, especially when squatting [fig_ref] Figure 1: The patient had difficulty in full squatting [/fig_ref]. She had difficulty in opening the mouth [fig_ref] Figure 1: The patient had difficulty in full squatting [/fig_ref]. The tongue movements, especially, lolling, were significantly compromised. Macroglossia with lateral scalloping was noticed [fig_ref] Figure 1: The patient had difficulty in full squatting [/fig_ref]. She also complained of the difficulty in swallowing and hoarseness when talking. Hardening in bilateral mandible areas were noticed. Muscle strength was roughly at the Grade 4. No evident edema was found in the lower limbs. Extensive laboratory examinations were performed and the relevant results were listed as follows: IgG: 8.68 g/L (7.51-15.6), IgM: 0.9 g/L (0.46-3.04), IgA: 0.86 g/L (0.82-4.53), free light kappa chain: 6.24 g/L (6.29-13.5), lambda chain 3.42 g/L (3.13-7.23), and blood calcium concentration was 2.59 mM/L. Common autoantibodies related to the rheumatic diseases were all negative. The concentration of complement 3 (C3) was slightly low while that of C4 was normal. A hardened nodule was found in submandibular area and ultrasound examination confirmed it was the thickened muscle in the base of the tongue. MRI examination of sublingual areas demonstrated abnormal signals in the bilateral epiglottis cartilage, laryngeal cavity, and the base of the tongue. The patient refused tongue biopsy. MRI of lower limbs showed a mild edema of subcutaneous soft tissue. Skeletal radiography did not detect apparent abnormality. Serum protein electrophoresis showed an apparent lambda light chain [fig_ref] Figure 2: Serum protein eletrophoresis showed an evident band of Lamda chain [/fig_ref]. Urine electrophoresis confirmed the existence of the Bence-Jones protein [fig_ref] Figure 2: Serum protein eletrophoresis showed an evident band of Lamda chain [/fig_ref]. Analysis of bone marrow specimens showed an increased percentage of plasma cells (12%) and immunostaining revealed a positive reactivity for a lambda light chain. Histological examination of skin biopsy [fig_ref] Figure 2: Serum protein eletrophoresis showed an evident band of Lamda chain [/fig_ref]. A diagnosis of AL amyloidosis with skin and muscle involvement was then established. The patient was then referred to the Department of Hematology at our university hospital where she received chemotherapy consisting of velcade, cyclophosphamide, and dexamethasone. After 2 rounds of chemotherapy, skin thickening and hardening and dysphagia were significantly alleviated. Consistently, serum protein electrophoresis showed a decreased band of the lambda chain, and flow cytometry demonstrated a decreased percentage of plasma cells (8%). # Discussion Monoclonal gammopathy (MG) of undetermined significance (MGUS) is an asymptomatic premalignant condition that can eventually progresses to a malignant plasma cell dyscrasia or lymphoproliferative disorder. MG with prominent skin involvements can be referred to MG of cutaneous significance (MGCS). [bib_ref] Monoclonal gammopathy of cutaneous significance: review of a relevant concept, Lipsker [/bib_ref] Here, we report a case of primary AL amyloidosis mainly manifested as significant extensive skin involvement and muscle lesion. The presentations were so atypical that the patient was misdiagnosed repeatedly as scleroderma even by the most experienced physicians in China. Macroglossia with slight lateral scalloping was an important sign that led us to investigate the possibility of AL amyloidosis. Other symptoms included difficulty in lolling and snoring, suggesting the pathologies in muscles. In addition, the difficulty in the movements of lower limbs, especially during squatting exercises, also indicates muscle involvement, although tightened skin may also play a role. AL amyloidosis results from the deposition of fibrillar protein consisting of the light chain of immunoglobulin with the lambda type accounting for 75% of all cases. [bib_ref] Dangerous small B-cell clones, Merlini [/bib_ref] While this clonal B-cell disorder may be associated with multiple myeloma (MM) and lymphoma, it can also be idiopathic. [bib_ref] Skin involvement in primary systemic amyloidosis, Kumar [/bib_ref] Clinical and laboratory data, for example, plasmacytosis (12%) from bone marrow assay, indicate that the original disease is (MM). Amyloidosis occurs in about 15% of MM patients.Protean manifestations often lead to delayed and incorrect diagnosis. Biopsy specimens with positive Congo red staining is critical for its diagnosis. Identification of the type of immunoglobulin light chains helps unveil the underlying diseases. [bib_ref] Unusual milia amyloidosis as initial signs of multiple myeloma-associated systemic amyloidosis, Ohashi [/bib_ref] Although cumbersome, mass spectrometry of amyloid material remains the gold standard for its final diagnosis. [bib_ref] Classification of amyloidosis by laser microdissection and mass spectrometry-based proteomic analysis in..., Vrana [/bib_ref] [bib_ref] Mass spectrometry-based proteomic diagnosis of renal immunoglobulin heavy chain amyloidosis, Sethi [/bib_ref] [bib_ref] Mass spectrometric-based proteomic analysis of amyloid neuropathy type in nerve tissue, Klein [/bib_ref] [bib_ref] Reliable typing of systemic amyloidoses through proteomic analysis of subcutaneous adipose tissue, Brambilla [/bib_ref] Cutaneous and mucous involvements have been reported by different research groups and the major manifestations are summarized in [fig_ref] Table 1: Skin, nail, and mucous manifestations [/fig_ref]. [bib_ref] Cutaneous amyloidoses and systemic amyloidoses with cutaneous involvement, Schreml [/bib_ref] Although scleroderma-like changes have also been reported before , [bib_ref] A case of amyloidosis due to multiple myeloma that resembled systemic sclerosis, Sabadini [/bib_ref] [bib_ref] Light chain multiple myeloma with peripheral leucocytosis presenting as scleroderma amyloidosum of..., Casper [/bib_ref] [bib_ref] Scleroderma-like manifestation in a patient with primary systemic amyloidosis: response to high-dose..., Cho [/bib_ref] [bib_ref] Nodular primary localized cutaneous amyloidosis after trauma: a case report and discussion..., Kalajian [/bib_ref] [bib_ref] Light chain multiple myeloma with cutaneous AL amyloidosis, Becker [/bib_ref] [bib_ref] Scleroderma-like illness as a presenting feature of multiple myeloma and amyloidosis, Reyes [/bib_ref] [bib_ref] Primary systemic (amyloid lightchain) amyloidosis masquerading as pseudoxanthoma elasticum: recognizing a novel..., Wat [/bib_ref] [bib_ref] Primary systemic amyloidosis initially presenting with digestive symptoms: a case report and..., Lin [/bib_ref] [bib_ref] AL amyloidoma of the skin/ subcutis: cutaneous amyloidosis, plasma cell dyscrasia or..., Walsh [/bib_ref] extensive cutaneous involvement seen in our patient is not common. Severe hardening and pigmentation led to an incorrect diagnosis as scleroderma repeatedly. After thorough examinations and discussions, we excluded the possibility of scleroderma because of the presence of the presence of microglossia and the absence of Raynaud phenomenon, and the anti-Scl-70 and ACA antibodies. In addition, low dose of glucocorticoid together with weekly use of methotrexate did not show any effect. The diagnosis of POEMS could not be established because of atypical clinical manifestations and the absence of evident angiogenesis up histological examination. After serum and urine electrophoresis and analysis of the skin and bone marrow specimens, a primary AL amyloidosis was established, which was consolidated by prompt response to chemotherapy. The treatment of AL amyloidosis is similar to that of MM, [bib_ref] Cyclophsphamide, thalidomide, and dexamethasone as induction therapy for newly diagnosed multiple myeloma..., Morgan [/bib_ref] with the ultimate goal being the eradication of monoclonal plasma cells in bone marrow and prevention of production of pathological immunoglobulin light chains. We hope that this report will help our peers keep vigilant of underlying diseases when similar clinical scenario occurs in order to make a prompt diagnosis and effective treatment. Scleroderma-like changes in primary amyloidosis. ## Ref. Journal Year Major findings Sabadini et al [bib_ref] A case of amyloidosis due to multiple myeloma that resembled systemic sclerosis, Sabadini [/bib_ref] J Rheumatol 1997 Scleroderma-like changes Casper et al [bib_ref] Light chain multiple myeloma with peripheral leucocytosis presenting as scleroderma amyloidosum of..., Casper [/bib_ref] Br J Dermatol 1999 peripheral leukocytosis Cho et al [bib_ref] Scleroderma-like manifestation in a patient with primary systemic amyloidosis: response to high-dose..., Cho [/bib_ref] Yonsei Med J 2006 cutaneous induration, dysphagia Kalajian et al [bib_ref] Nodular primary localized cutaneous amyloidosis after trauma: a case report and discussion..., Kalajian [/bib_ref] J Am Acad Dermatol 2007 Trauma induced nodular cutaneous amyloidosis Becker et al [bib_ref] Light chain multiple myeloma with cutaneous AL amyloidosis, Becker [/bib_ref] J Dtsch Dermatol Ges 2008 Painful sclerotic skin changes Reyes et al [bib_ref] Scleroderma-like illness as a presenting feature of multiple myeloma and amyloidosis, Reyes [/bib_ref] J Clin Rheumatol 2008 Carpal tunnel syndrome, polyarthritis, thickened skin, dysphagia, ecchymotic rashes Wat et al [bib_ref] Primary systemic (amyloid lightchain) amyloidosis masquerading as pseudoxanthoma elasticum: recognizing a novel..., Wat [/bib_ref] JAMA Dermatol 2014 Pseudoxanthoma elasticum Lin et al [bib_ref] Primary systemic amyloidosis initially presenting with digestive symptoms: a case report and..., Lin [/bib_ref] Diagn Pathol gastrointestinal involvement, periorbital purpura, petechiae, ecchymoses and sclerosis Walsh et al [bib_ref] AL amyloidoma of the skin/ subcutis: cutaneous amyloidosis, plasma cell dyscrasia or..., Walsh [/bib_ref] Am J Surg Pathol Cutaneous and subcutaneous induration, dysphagia [fig] Figure 2: Serum protein eletrophoresis showed an evident band of Lamda chain (L) of immunoglobulin (A). The Bence-Jones Protein (BJP) was detected after urine electrophoresis (B). Histological examinations of skin biopsy specimens showed amorphous, Congo-red positive deposits (arrow) in the dermis (C). [/fig] [fig] Figure 1: The patient had difficulty in full squatting (A) and mouth opening (B). The movements of tongue were limited, especially the extension. Macroglossia with slight lateral scalloping was noticed ( [/fig] [table] Table 1: Skin, nail, and mucous manifestations. Skin Smooth and hyperpigmented papules and papules Hemorrhagic bullae and dissecting hematomas Scleroderma-like changes Cutis laxa and blue skin tint Ecchymosis and purpura [/table]

Directional local field potentials: A tool to optimize deep brain stimulation [bib_ref] Directional deep brain stimulation: an intraoperative double-blind pilot study, Pollo [/bib_ref] [bib_ref] Multiple-source current steering in subthalamic nucleus deep brain stimulation for Parkinson's disease..., Timmermann [/bib_ref] [fig_ref] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy [/fig_ref] [bib_ref] Basic algorithms for the programming of deep brain stimulation in Parkinson's disease, Volkmann [/bib_ref] [bib_ref] Programming deep brain stimulation for Parkinson's disease: The Toronto Western Hospital Algorithms, Picillo [/bib_ref] [bib_ref] Subthalamic nucleus stimulation: improvements in outcome with reprogramming, Moro [/bib_ref] [fig_ref] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy [/fig_ref] [bib_ref] Innovations in deep brain stimulation methodology, K€ Uhn [/bib_ref] [bib_ref] Intra-operative recordings of local field potentials can help localize the subthalamic nucleus..., Chen [/bib_ref] [bib_ref] Subthalamic span of beta oscillations predicts deep brain stimulation efficacy for patients..., Zaidel [/bib_ref] [bib_ref] Subthalamic span of beta oscillations predicts deep brain stimulation efficacy for patients..., Zaidel [/bib_ref] [bib_ref] Value of subthalamic nucleus local field potentials recordings in predicting stimulation parameters..., Yoshida [/bib_ref] [bib_ref] Guiding deep brain stimulation contact selection using local field potentials sensed by..., Connolly [/bib_ref] # Patients and methods Twelve PD patients undergoing STN-DBS surgery were implanted with directional leads (Boston Scientific, Marlborough, MA; Supplementary . LFPs were recorded during surgery from the directional contacts after each lead was placed in its final position [fig_ref] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy [/fig_ref]. Normalized beta activity was derived from each directional contact by normalization of the individual beta peak activity by the whole beta band . In cases where no beta peak was present, the low beta band (13-20 Hz) was normalized. Monopolar contact review took place 4 to 7 months postsurgery. Clinical efficacy (% rigidity improvement/stimulation current) and therapeutic window (TW) were calculated for each directional contact and compared with the corresponding normalized beta activity. Detailed methods are included in the Supplementary Material. # Results ## Relationship between beta activity and response to stimulation In 15 of 19 hemispheres tested, we found a positive relationship (t 18 5 4.65; P < 0.001, one-sample t test) between normalized beta activity and clinical efficacy (ranked values: [fig_ref] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy [/fig_ref] ; absolute nonranked values: . Thus, the higher the relative beta activity recorded from a specific directional contact, the better its clinical efficacy. In all cases, the contact with highest beta was consistently one of those with higher clinical efficacy, and in 12 of 19 cases (63%), it corresponded to the contact with the highest clinical efficacy. In 7 of 19 cases (36%; hemispheres 13-19), we did not find a clear beta peak, but despite this, the relationship was similar to those with a clear peak in the beta band. There was also no difference in the predictive value of the level and orientation of the beta peak between those hemispheres with a beta peak up to 20 Hz (52, 3, 5, 6, 7, 10, and 11) and those hemispheres with a beta peak above 20 Hz (hemispheres . The ring level ## Predictive value of beta activity for the most efficient stimulation contact In [fig_ref] FIG. 2: LFP-based DBS programming [/fig_ref] , we tested the predictive value of contacts ranked by relative beta power for clinical efficacy. This shows that the stimulation contact with the highest beta activity was able to predict the stimulation contact with the highest clinical efficacy in 63% of cases. More strikingly, when including the contact with the second-highest beta activity, the prediction rose to 84%, and up to 92% if only hemispheres with a clear beta peak (n 5 12) were considered. In contrast, conventional clinical testing had only a 17% likelihood of identifying the most efficient contact if only one contact was assessed, and a 34% likelihood if two contacts were assessed. [fig_ref] FIG. 2: LFP-based DBS programming [/fig_ref] shows that the mean clinical efficacy of the two contacts with the highest beta activity was significantly higher (31.3 6 3.2%/mA [milliamperes]) compared to the mean clinical efficacy (26.1 6 2.7%/mA) of the remaining contacts of the same electrode (t 18 5 3.75; P 5 0.0015, paired t test). ## Relationship between clinical efficacy and therapeutic window Another important clinical parameter is the therapeutic window, which also includes the side-effect threshold. [fig_ref] FIG. 2: LFP-based DBS programming [/fig_ref] shows that the LFP-based strategy identified the contact with the widest therapeutic window in 42% of cases if only the contact with the highest beta activity was considered, and in 74% if the two highest beta contacts were considered. No relevant difference in the predictive value was found when hemispheres with a clear beta peak were exclusively considered. Additionally, [fig_ref] FIG. 2: LFP-based DBS programming [/fig_ref] shows that the mean therapeutic window of the two contacts with the highest beta activity was significantly higher (1.45 6 0.27 mA) compared to the mean TW (0.96 6 0.17 mA) of the remaining contacts of the same electrode (t 18 5 3.11; P 5 0.006, paired t test). # Discussion In this study, we demonstrate, in a sizeable patient cohort, that the two segmented contacts of the directional DBS electrode with maximal STN beta activity are highly likely to include the contact that turns out to have the best efficacy with a wide therapeutic window. Clinical testing was performed at least 4 months after lead implantation, when the majority of any stun effect has lapsed 16 and the clinical relevance of contact screening therefore heightened. Thus, the LFP can serve as a predictive and supportive tool for multicontact lead programming. This is in line with previous studies showing similar results for the ring contact electrode, [bib_ref] Subthalamic span of beta oscillations predicts deep brain stimulation efficacy for patients..., Zaidel [/bib_ref] [bib_ref] Value of subthalamic nucleus local field potentials recordings in predicting stimulation parameters..., Yoshida [/bib_ref] [bib_ref] Guiding deep brain stimulation contact selection using local field potentials sensed by..., Connolly [/bib_ref] as well as with an intraoperative trial [bib_ref] Directional recording of subthalamic spectral power densities in Parkinson's disease and the..., Bour [/bib_ref] and a single, early postoperative case report with directional stimulation. [bib_ref] Directional local field potential recordings for symptom-specific optimization of deep brain stimulation, Fern Andez-Garc Ia [/bib_ref] Why should beta power in the LFP predict the clinical efficacy of stimulation fields of different orientation? It has been shown that the dorsal part of STN is the most effective site for STN stimulation in PD, [bib_ref] Most effective stimulation site in subthalamic deep brain stimulation for Parkinson's disease, Herzog [/bib_ref] [bib_ref] Localization of electrodes in the subthalamic nucleus on magnetic resonance imaging, Pollo [/bib_ref] and that this is also the focus of beta activity. [bib_ref] Subthalamic span of beta oscillations predicts deep brain stimulation efficacy for patients..., Zaidel [/bib_ref] [bib_ref] Value of subthalamic nucleus local field potentials recordings in predicting stimulation parameters..., Yoshida [/bib_ref] Yet, the LFP cannot afford direct information about the contact specific therapeutic window, because side effect threshold depends on the vicinity of the stimulation field to neighboring structures. On the other hand, as current directed to the dorsal STN is less likely to spread to these neighboring areas, the prediction of the contact with the lowest threshold for clinical effect may also explain the predictive value for the contact with the widest therapeutic window. ## Lfp-based programming If we assume that it takes around 20 minutes to assess stimulation at each contact, then monopolar contact review of segmented leads will take around 4 hours (12 segmented contacts). This would be fatiguing for both clinician and patient, leading to variability in assessments. If only those two segmented contacts that have the highest beta activity are screened on each side, then there is approximately a 90% probability of selecting the contacts that have the lowest effect threshold. This would only take 80 minutes, reducing assessment time by approximately two thirds. Moreover, as discussed . Contacts are distributed along four levels. On levels two and three, there are three segmented contacts (level two: contacts 2/3/4; level three: contacts 5/6/7). (B) shows an example time frequency spectrum from an intraoperative LFP recording (duration, 100 seconds) from the six directional contacts (2/3/4; 5/6/7) with the patient awake and at rest. The dashed white line marks the beta frequency band . It shows that LFP beta activity is not equally distributed across directional contacts. Contact 5 shows the highest beta activity, followed by contact 2, with both contacts 5 and 2 oriented in the same direction. Data from the right hemisphere in subject 3 (for raw data, amplitude-frequency spectrum, and imaging from the same subject and hemisphere, see [fig_ref] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy [/fig_ref]. (C) illustrates the relationships between normalized beta activity and clinical efficacy across the six directional contacts in each hemisphere (H 5 hemisphere; n . The normalized beta amplitude is shown on the x-axis, the clinical efficacy on the y-axis, and Spearman correlation coefficients are shown on the top of each panel. The best electrophysiological contact (contact with highest normalized beta activity) is highlighted in black. The red linear regression fit is shown only for illustration purposes. In 15 hemispheres, a positive relationship between clinical efficacy and normalized beta activity was found [bib_ref] Directional local field potential recordings for symptom-specific optimization of deep brain stimulation, Fern Andez-Garc Ia [/bib_ref] above, the two segmented contacts with the highest beta activity are also more likely to have a wider therapeutic window. Hence, this method potentially offers a physiologically based, time-saving approach to the programming of directional electrodes. # Limitations In this investigation, we only studied those sides with at least two points of upper-limb rigidity and more than a minimum range of responses to stimulation across contacts. We also limited the electrophysiological-clinical comparison to the clinical data acquired during the monopolar review session, where rigidity was the only systematically assessed item. However, rigidity is also the most sensitive clinical sign to DBS. [bib_ref] Basic algorithms for the programming of deep brain stimulation in Parkinson's disease, Volkmann [/bib_ref] These inclusion criteria were chosen to optimize the clinical comparison across contacts, and to avoid ceiling and floor effects. The value of beta activity and of other LFP features in predicting the best contact for tremor suppression needs further evaluation. In addition, we assumed that the monopolar review in and of itself is predictive of chronic stimulation settings. [bib_ref] Basic algorithms for the programming of deep brain stimulation in Parkinson's disease, Volkmann [/bib_ref] [bib_ref] Most effective stimulation site in subthalamic deep brain stimulation for Parkinson's disease, Herzog [/bib_ref] Moreover, manual clinical contact testing, although the current "gold standard" for determining the best stimulation contacts, is a subjective method with some degree of inter-rater variability. Any noise in the gold-standard estimation will only have served to degrade the apparent predictive value of the LFP. Intraoperative time constraints meant that LFPs could only be recorded for around 2 minutes (with interindividual variability), and longer recordings might have been more representative. Importantly, we also assumed that lead position and orientation did not change after LFP recording. Furthermore, our data may have been contaminated by stun effects, which can be detected as the STN is traversed and lead to diminished beta power. [bib_ref] Intra-operative recordings of local field potentials can help localize the subthalamic nucleus..., Chen [/bib_ref] ## Future directions and conclusion Tools that can assist DBS programming by the clinician or even run fully automatically are desirable in this era of directional, multicontact leads. This could streamline the postoperative management of patients, and free up clinical resources to contend with the increasing numbers of such patients dictated by growing experience with this therapy and by the move to offer DBS earlier during the disease course. [bib_ref] Neurostimulation for Parkinson's disease with early motor complications, Schuepbach [/bib_ref] Nevertheless, the clinical advantage, or lack thereof, of chronic directional DBS still needs to be definitively demonstrated. The method presented is of potential predictive value with respect to subsequent programming, regardless of whether microelectrode recordings are used or not in targeting the STN. In time, optimal contact prediction might be based on a variety of features. The electrophysiological approach taken here might be supplemented by radiological-anatomical strategies that could provide more-accurate information about surrounding structures. However, presently, these are challenging to implement, given that target structures are small and image resolution is limited, so that the error rate in lead localization is not negligible. [bib_ref] Coordinate-based lead location does not predict Parkinson's disease deep brain stimulation outcome, Nestor [/bib_ref] In conclusion, the present study suggests that the amplitude of subthalamic beta LFP activity is predictive of the most efficient stimulation contact and can form the basis for a rapid programming tool useful for multicontact directional DBS leads. [fig] FIG. 1: Directional LFPs and relationship between ranked beta activity and clinical efficacy. (A) illustrates the directional DBS lead (Boston Scientific, Marlborough, MA) [/fig] [fig] 5: 4.65; P < 0.001, one-sample t test). In 12 of 19 hemispheres, the contact with the highest beta activity matched the clinically most effective stimulation contact. Furthermore, in all hemispheres, the contact with the highest beta activity was localized in the upper-right quadrant, where the clinically more efficient contacts are localized. Clinical efficacy and normalized beta activity are illustrated as ranked values; Supplementary Figure 3 shows the same figure with nonranked values. [Color figure can be viewed at wileyonlinelibrary.com] [/fig] [fig] FIG. 2: LFP-based DBS programming. (A) shows the probability of identifying the stimulation contact with the highest clinical efficacy, comparing the conventional (random) test strategy in blue with the LFP-based test strategy in red (full red line: all hemispheres n 5 19, dashed red line: only hemispheres with clear beta peak n 5 12). While for conventional mapping the probability of identifying the most efficient stimulation contact increases by 0.17 with each contact tested, the LFP-based strategy identifies the most efficient contact with a probability of 0.63 if only the contact with the highest beta activity is considered, and with a probability of 0.84 if the two contacts with the highest beta activity are considered. By considering hemispheres with a clear beta peak only, the probability increases up to 0.92 when the two best electrophysiological contacts are considered. (B) The mean clinical efficacy of the two directional stimulation contacts with the highest beta activity ("Best ephys") is significantly higher compared to the clinical efficacy of the remaining directional contacts ("other dir."). (C) (similar to A) shows the probability of identifying the contact with the highest therapeutic window by again comparing the conventional (random) test strategy in blue with the LFP-based test strategy in red (full red line: all hemispheres n 5 19; dashed red line: only hemispheres with clear beta peak n 5 12). While for conventional mapping the probability of identifying the stimulation contact with the widest therapeutic window increases by 0.17 with each contact tested, the LFP-based strategy identifies the contact with the widest therapeutic window with a probability of 0.42 if only the contact with the highest beta activity is considered, and with a probability of 0.74 if the two contacts with the highest beta activity are considered. No relevant difference in the predictive value is found when exclusively hemispheres with a clear beta peak are considered (dashed red line). (D) The mean TW of the two directional stimulation contacts with the highest beta activity ("Best ephys") is significantly higher compared to the therapeutic window of the remaining directional contacts. Values are mean 6 SEM; **P < 0.01. [Color figure can be viewed at wileyonlinelibrary. [/fig] [table] 32: Savica R, Grossardt BR, Bower JH, Ahlskog JE, Rocca WA. Time trends in the incidence of Parkinson disease. Normalized beta activity was positively correlated with the contact's clinical efficacy. The two contacts with the highest beta activity included the most efficient stimulation contact in up to 92% and that with the widest therapeutic window in 74% of cases. Conclusion: Local field potentials predict the most efficient stimulation contacts and may provide a useful tool to expedite the selection of the optimal contact for directional DBS.V C 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. [/table]

Effect of Genomic and Amino Acid Sequence Mutation on Virulence and Therapeutic Target of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS COV-2) The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is one of the RNA coronaviruses which share the highest mutation rates of RNA viruses when compared with that of their hosts. The collective mutation rate of RNA viruses is up to a million times higher than their hosts and is correlated with enhanced virulence of viruses. The RNA, genomic material of SARS-CoV-2, has the capacity of showing amplified fast changes as the infection spreads. These changes were frequently observed in genes for spike glycoprotein, nucleocapsid, ORF1ab, and ORF8, together with RNA dependent RNA polymerase. In contrast, genes for envelope, membrane, ORF6, ORF7a and ORF7b showed no observable changes in terms of amino acid substitutions. Mutated SARS COV-2 at these particular sites has been associated with viral infectivity, false laboratory results and viral genome mutation and interferes with therapeutic targets. Interferences with therapeutic targets is frequently observed in genes for RdRp. Additionally, mutated viral genes for RdRp render slow fidelity of RdRp protein, resulting in a high mutation rate. Such a high mutation rate might allow new virulent forms of the virus to emerge and influence the disease profile. This review aimed to elaborate on the effect of genomic and amino acid sequence mutations on the virulence and therapeutic targets of SARS COV-2. To achieve this objective, multiple literatures have been reviewed. # Introduction # Background information Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA coronavirus belonging to genus β-coronavirus and a member of the family of Coronaviridae. [bib_ref] A novel coronavirus from patients with pneumonia in China, Ma [/bib_ref] Most Coronaviridae families, including those causing respiratory problems in human hosts, are belonging to two genera, α-coronavirus and β-coronavirus. The extent of respiratory problems differs from person to person. For instances, it causes mild infection in healthy adults but has much more severe effects in the elderly and patients with co-morbidities. The later can experience severe damage to their respiratory systems, sometimes dying as a result. [bib_ref] Likelihood of survival of coronavirus disease 2019 Scientific and ethical basis for..., Ruan [/bib_ref] Several literatures indicated that people older than 65 years and those with underlying co-morbidities experience higher mortality rates than former ones. [bib_ref] Likelihood of survival of coronavirus disease 2019 Scientific and ethical basis for..., Ruan [/bib_ref] [bib_ref] A novel coronavirus outbreak of global health concern, Wang [/bib_ref] [bib_ref] Variant analysis of SARS-CoV-2 genomes, Koyama [/bib_ref] [bib_ref] Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in..., Richardson [/bib_ref] [bib_ref] Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia..., Chen [/bib_ref] [bib_ref] Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in..., Yang [/bib_ref] Fever, cough, shortness of breath and fatigue are common symptoms of the coronavirus disease , caused by SARS COV-2. [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Guan [/bib_ref] [bib_ref] Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in..., Wang [/bib_ref] As indicated in early data, about 20% of patients developed severe symptoms of COVID-19 accompanied by shortness of breath and requiring hospitalization, including 5% who are admitted to intensive care units. Besides, the initial estimate of case fatality rates from COVID-19 was from 3.4% to 6.6%, which was much extermely lower than the 9.6% for severe acute respiratory syndrome (SARS) or the 34.3% for Middle East respiratory syndrome (MERS). [bib_ref] A novel coronavirus outbreak of global health concern, Wang [/bib_ref] [bib_ref] Variant analysis of SARS-CoV-2 genomes, Koyama [/bib_ref] Regardless of the fact that the outbreak has dramatically expanded to create a global pandemic, 8 the COVID-19 related morbidity, numbers of active cases, and mortality rates differ from country to country. Many studies from different corners of the world speculating on possible reasons for these variations but have produced no credible results, yet. According to different studies from different corners of the globe, the 4715L variant of open reading frame 1ab (ORF1ab) protein, 614G variant of S protein and RNA dependent RNA polymerase (RdRp) were the commonest viral proteins affected by mutations among others. [bib_ref] SARS-CoV-2 genomic variations associated with mortality rate of, Toyoshima [/bib_ref] [bib_ref] Emerging SARS -CoV -2 mutation hot spots include a novel RNA -dependent..., Pachetti [/bib_ref] One study hypothesized that there is a correlation between mutations at the 4715L variant of ORF1ab and 614G variant of S protein and fatality rates. [bib_ref] SARS-CoV-2 genomic variations associated with mortality rate of, Toyoshima [/bib_ref] Moreover, mutated RdRp may allow the viruses to escape host immunity and to develop resistance to commonly used antiviral therapies. [bib_ref] Emerging SARS -CoV -2 mutation hot spots include a novel RNA -dependent..., Pachetti [/bib_ref] Therefore, dealing with the effects of mutation on virulence and therapeutic targets of this virus should be needed to overcome such challenges, hence, the aim of this review was to shed light on the effects of genes and amino acid sequence mutation on virulence and therapeutic targets of SARS COV-2. ## Sars cov-2 genome and genomic expression SARS-CoV-2 has a single strand RNA genome, which is positive-sensed and about 29,903 kb nucleotides long, making it as one of the largest genomes among RNA viruses. 14 These nucleotides are organized into 10 ORFgenes encoding for 28 proteins. Four out of 28 proteins are structural proteins (spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins), 16 are nonstructural proteins (Nsps) and 8 are accessory proteins. [bib_ref] Genotype and phenotype of COVID-19: their roles in pathogenesis, Mousavizadeh [/bib_ref] [bib_ref] A new coronavirus associated with human respiratory disease in China, Tao [/bib_ref] [bib_ref] A pneumonia outbreak associated with a new coronavirus of probable bat origin, Zhou [/bib_ref] [bib_ref] The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19),..., Yoshimoto [/bib_ref] The spike (S) glycoprotein is comprised 1273 amino acid residues and is critical for the viral infection. It has N-terminus signal peptide of 1-13 amino acid residues, S1 subunit peptide of 14-685 amino acid residues, and S2 sub unit peptide of 686-1273 amino acid residues. [bib_ref] Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development..., Huang [/bib_ref] [bib_ref] One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase..., Subissi [/bib_ref] [bib_ref] Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and..., Jia [/bib_ref] The S1 subunit peptide has N-terminal domain (composed of 14-305 stretch of amino acid residues) and a receptor-binding domain (RBD) (composed of 319-541 stretch of amino acid residues. The S2 subunit peptide has a fusion peptide (FP) (composed of 788-806 stretch of amino acid residues), a heptapeptide repeat sequence 1 (HR1) (composed of 912-984 stretch of amino acid residues), an HR2 (composed of a 1163-1213 stretch of amino acid residues), a transmembrane domain (composed of 1213-1237 stretch of amino acid residues), and a cytoplasmic domain (composed of 1237-1273 stretch of amino acid residues). [bib_ref] Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development..., Huang [/bib_ref] The virus to host interaction is initiated when S glycoprotein, through its RBD of S1 subunit peptide, interacts with angiotensin-converting enzyme-2 (ACE-2) receptor on the host cell surface. The RBD-ACE-2 interaction allows not only viral infection, but also enhances rapid human-to-human transmission. [bib_ref] One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase..., Subissi [/bib_ref] Mutation of these sitesdo have effect on viral virulence.The other site on the viral genome prone to mutation is ORF1ab, encoding for two viral polypeptides (Pp1a and Pp1b), which generate 16Nsps after cleavage by chymotrypsin-like (3CL) protease (M pro ) and papain-like proteases. [bib_ref] One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase..., Subissi [/bib_ref] [bib_ref] Infection, genetics and evolution genetic diversity and evolution of SARS-CoV-2, Phan [/bib_ref] [bib_ref] The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19),..., Yoshimoto [/bib_ref] Gene and Amino Acid Mutations of SARS COV-2 Gene Mutations RNA viruses overwhelms their immediate hosts in mutation rates which was highly correlated with enhanced virulence of virus. [bib_ref] Why are RNA virus mutation rates so damn high?, Duffy [/bib_ref] The SARS-CoV-2 genome showed increasing fast and frequent changes at the level of genes for S, N, ORF1ab ORF8 proteins as the spread of infection amplified. [bib_ref] Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins..., Lu [/bib_ref] Study done on six geographic regions by taking SARS-CoV-2 as the reference identified a total of 1234 mutations. [bib_ref] SARS-CoV-2 genomic variations associated with mortality rate of, Toyoshima [/bib_ref] Another study done in USA analyzed the rate of accumulated viral gene mutations and identified accumulated mutations on genes for helicase, NSP2, S, NSP3,RdRp, ORF3a, ORF8, and N proteins. among others.Similarly, R203K and G204R mutations are commonly identified at the level of N proteins. [bib_ref] Evolutionary dynamics of SARS-CoV-2 nucleocapsid protein (N protein) and its consequences, Rahman [/bib_ref] There are no mutations identified yet in relation to M and E proteins. The possible reason might be housekeeping functions of these proteins allowing them to have a greater resistance to mutations.Amino Acid Mutations for Non-Structural Proteins ORF3a, N, ORF8, ORF1ab, and S proteins showed the largest number of non-synonymous mutations, with ORF3a taking the leading position. [bib_ref] Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could..., Benvenuto [/bib_ref] The Q57H and G251V mutations account for the majority of nonsynonymous mutations identified in ORF3a. Similarly, L84S is a major non-synonymous mutation in ORF8, accompanied by a change in V62L.Comparable levels of mutation frequencies have been observed at the level of NSP2, NSP3 and RdRp proteins with that of ORF3a, N, ORF8, ORF1ab and S proteins.The nsp2 (Asp268del) deletion mutation is the most common and widely spread type of non-synonymous mutation across Europe. [bib_ref] Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals..., Bal [/bib_ref] Like E and M structural proteins, ORF6, 7a, 7b and 10, NSP7, NSP9, NSP10, and NSP11 proteins showed no significant changes in amino acid residues, yet. [bib_ref] Evolutionary and structural analyses of SARS -CoV -2 D614G spike protein mutation..., Isabel [/bib_ref] The 27-amino-acid deletion in ORF7a SARS-CoV-2 reported in Arizona was exceptional. 56 ## Impact of orf1ab gene mutation in laboratory results and amino acid sequences of encoded proteins ORF1ab occupies more than 75% of the SARS COV-2 genome and is identified as the highest mutation frequency region. [bib_ref] Mutational frequencies of SARS-CoV-2 genome during the beginning months of the outbreak..., Kaushal [/bib_ref] [bib_ref] Analysis of SARS-CoV-2 RNA-dependent RNA polymerase as a potential therapeutic drug target..., Aftab [/bib_ref] In Japan, three and twenty-four nucleotide deletion mutations were identified within this region, and in Australia, twenty-nine mis-sense mutations were identified in the ORF1ab polyprotein. [bib_ref] Infection, genetics and evolution genetic diversity and evolution of SARS-CoV-2, Phan [/bib_ref] False positive laboratory result, as it permit infected individuals to live with other people and, might have an unfavorable impacts on endeavors to control the spread of the virus. [bib_ref] A recurrent mutation at position 26,340 of SARS-CoV-2 is associated with failure..., Maria [/bib_ref] Currently accepted laboratory test for SARS-CoV-2, the real-time polymerase chain reaction (RT-PCR) test, risks producing inconsistent results. The possible reasons for this inconsistency might be mutations in viral RNA sequences affecting RT-PCR-utilizing primers in different genes; or mutations in the primer and probe target regions; or genetic diversity and rapid evolution of SARS-CoV-2 that have been observed in different studies. [bib_ref] Real-time RT-PCR in COVID-19 detection: issues affecting the results, Tahamtan [/bib_ref] The 14,408C>T (P4715L) and 3037 C>T (F106F) variants have been recognized as the two most frequently occurring gene mutations at ORF1ab regions, possibly leading to inconsistent RT-PCR test results. The P4715L variant was causing mutations in the RdRp/NSP12 and responsible for lower replication fidelity of the enzyme. The F106F variant, on the other hand, was causing mutations in the Nsp3 gene. [bib_ref] Identification of novel mutations in SARS-COV-2 isolates from, Rehman [/bib_ref] Furthermore, ORF1ab regions of the virus from different geographic regions showed mutation of amino acid sequences of encoded proteins. An isolate from China showed N2708S and F2908I, whereas from South Korea and Sweden showed G818S and F4321L. On the other hand, an isolate from Brazil showed L3606F and from Vietnam showed R3323C. [bib_ref] Comparative genome analysis of novel coronavirus (SARS-CoV-2) from different geographical locations and..., Khan [/bib_ref] [bib_ref] Drug Resistance is an international, peer-reviewed openaccess journal that focuses on the..., Benedetti [/bib_ref] ## Effect of mutant spike protein on virulence of sars cov-2 The affinity of RBD of the S sub-unit peptide of the S protein for ACE-2 and the overall infectivity of the virus has been potentially altered by gene mutations for spike. [bib_ref] Infection, genetics and evolution genetic diversity and evolution of SARS-CoV-2, Phan [/bib_ref] [bib_ref] Vulnerabilities in coronavirus glycan shields despite extensive glycosylation, Watanabe [/bib_ref] The overall impact is either reduced [bib_ref] Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and..., Jia [/bib_ref] or enhanced virulence of the virus depending on the site of mutation. [bib_ref] D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity, Hu [/bib_ref] [bib_ref] Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural stability..., Ou [/bib_ref] [bib_ref] Evaluating the effects of SARS-CoV-2 Spike mutation D614G on transmissibility and pathogenicity, Volz [/bib_ref] [bib_ref] Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated..., Eaaswarkhanth [/bib_ref] Aspartate to glycine mutation (D614G) of the S protein was thought to be 10-times more virulent than the original strain from China (Wuhan-1). [bib_ref] D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity, Hu [/bib_ref] Patients infected by the D614G mutant variant had advanced viral loads, [bib_ref] Spike mutation pipeline reveals the emergence of a more transmissible form of..., Korber [/bib_ref] and the overall disease progress was severe when compared with original China (Wuhan-1) strain-infected patients.The possible explanation might be related to the higher affinity D614G mutant strain for ACE2 than the original China (Wuhan-1) strain.Furthermore, the potential electro-static change from polar-aspartate to non-polar-glycine on the surface of D614G mutant S protein creates a favorable environment for mutant variants in a hydrophobic pocket of the S protein. Moreover, the change from large-aspartate residue to small glycine residue on the D614G mutant variant might increase the flexibility of a smooth switchover from the inactive DOWN state to the active UP state. [bib_ref] D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity, Hu [/bib_ref] [bib_ref] Evaluating the effects of SARS-CoV-2 Spike mutation D614G on transmissibility and pathogenicity, Volz [/bib_ref] Additionally, D614G facilitates cleavage of the spike protein by host proteases and thereby enhances membrane fusion, which is critical for viral entry into host cells. [bib_ref] D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity, Hu [/bib_ref] [bib_ref] Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated..., Eaaswarkhanth [/bib_ref] Similar to D614G, V367F, W436R, and D364Y are three other mutant variants of S protein emerging in Wuhan, Shenzhen, Hong Kong, and France. These variants have DovePress enhanced structural stabilization of the RBD beta-sheet scaffold and also displayed a higher affinity for human ACE2 just like to D614G variant of the S protein. [bib_ref] Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural stability..., Ou [/bib_ref] In contrast to D614G and other mutations which enhance infectivity of the virus, there are also mutations which reduce it, such as glycosylation mutants (N331 and N343) mediating protein folding and stability, and amino acid mutation in RBD (R408I). Glycosylation and RBD mutants together lead to drastically reduced viral infectivity. [bib_ref] Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and..., Jia [/bib_ref] [bib_ref] Pro fi ling and characterization of SARS-CoV-2 mutants ' infectivity and antigenicity, Wang [/bib_ref] [bib_ref] Variations in SARS-CoV-2 spike protein cell epitopes and glycosylation profiles during global..., Xu [/bib_ref] R408I mutation seems to disrupt the 408R-glycan hydrogen bond by changing hydrophilic arginine residue with hydrophobic isoleucine residue with no hydrogenbond potential. As evidence for reduced viral infectivity, potentially reduced ACE2 binding affinity which led to reduced virulence of the virus was observed with the R408I mutant strain identified in India. 20 ## Effect of mutant rdrp on gene mutation and therapeutic target ViralRdRp, also known as NSP12, is the complex protein with multiple domains that performs viral RNA synthesis and genome duplication. [bib_ref] Identi fi cation of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2..., Chand [/bib_ref] An RdRp mutation, particularly in position 14408, makes a great contribution to overall increased viral genome mutation rates. There are also reports on the mutation of P323L in RdRp, [bib_ref] Emerging SARS -CoV -2 mutation hot spots include a novel RNA -dependent..., Pachetti [/bib_ref] [bib_ref] Identification of novel mutations in SARS-COV-2 isolates from, Rehman [/bib_ref] which could resulted in structural rigidity of the protein and consequently lower replication fidelity of RdRp.Lower fidelity of RdRp also leads to higher mutation rates of the virus within host cells, which in turn allows new virulent forms to emerge and influence disease profile. [bib_ref] RdRp mutations are associated with SARS-CoV-2 genome evolution, Karakülah [/bib_ref] Moreover, mutant SARS COV-2 at P323L in RdRp has made infected COVID-19 patients to develop much more severe symptoms.In addition to its contribution to increased viral genomic mutation rates, mutant RdRp might affect the efficacy of antiviral drugs and thereby disturb therapeutic targets. Antiviral drugs such as remdesivir via their active component (adenosine nucleotide analog) binding to the active sites of RdRpand are being considered potential therapeutic options through viral replication inhibition. [bib_ref] Analysis of SARS-CoV-2 RNA-dependent RNA polymerase as a potential therapeutic drug target..., Aftab [/bib_ref] [bib_ref] Identi fi cation of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2..., Chand [/bib_ref] [bib_ref] RNA-dependent RNA polymerase of SARS-CoV-2 as a therapeutic target, Wang [/bib_ref] The active component of remdesivir binds to the RdRp catalytic site and halts nucleic acid elongation. Binding affinity with remdesivir profoundly decreased in several mutants, particularly F480L and V557L variants, strongly suggesting drug resistance. [bib_ref] Rational design of the remdesivir binding site in the RNA-dependent RNA polymerase..., Padhi [/bib_ref] [bib_ref] Coronavirus susceptibility to the antiviral remdesivir(GS-5734) is mediated by the viral polymerase..., Agostini [/bib_ref] Mutation of RdRpat A97V, A185V and P323 causes alteration of the secondary structure of RdRp. In mutation of RdRp at A97V and A185V, valine (with a relatively larger side chain than alanine) substitutes the alanine (with a relatively smaller side chain than valine) amino acid. In mutation of RdRp at P323L, the proline (with a bulky pyrrolidine ring side chain) substitutes by leucine that might result in loss of the structural integrity of the protein. [bib_ref] Identi fi cation of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2..., Chand [/bib_ref] [bib_ref] RdRp mutations are associated with SARS-CoV-2 genome evolution, Karakülah [/bib_ref] Recent studies on virtual molecular docking identified many drugs as SARS-CoV-2 RdRp inhibitors, including Simeprevir, Filibuvir, andTegobuvir. [bib_ref] RNA-dependent RNA polymerase of SARS-CoV-2 as a therapeutic target, Wang [/bib_ref] The P323L and L329I mutations of RdRp were identified very close to the docking site, probably interfering with the interaction of these drugs with RdRp. [bib_ref] Identi fi cation of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2..., Chand [/bib_ref] [bib_ref] RNA-dependent RNA polymerase of SARS-CoV-2 as a therapeutic target, Wang [/bib_ref] [bib_ref] The mechanism of resistance to favipiravir in influenza, Goldhill [/bib_ref] [bib_ref] Identification of a novel resistance mutation for benzimidazole inhibitors of the HCV..., Delang [/bib_ref] # Conclusion The causative agent of the COVID-19 pandemic is prone to a high mutation rate, which leads to changes in the properties of the virus, including its virulence or infectivity, and resistance to antiviral drugs. For instance, mutated SARS COV-2 in S protein at D614G infects ACE2 expressing cells more efficiently, and these boost its virulence. Thus, a patient infected by a mutant variant has higher viral loads and develops more severe symptoms of COVID-19. Mutated SARS COV-2 at RdRp regions causes low fidelity of RdRp, resulting in higher mutation rates, which in turn lead to the emergence of new virulent strains, influence overall disease profile and interfere with RdRp-targeting pharmacological agents. Even though a number of genome sequences of this virus have been identified and published, by no means all countries have sequenced the viral genome as yet, information which is important for recognizing mutated genes and newly emerging virus properties. # Copyright/ethical concerns Since this is a review article, there are no ethical concerns related to it. # Disclosure The authors report no conflicts of interest in this work.

Our emerging understanding of the roles of long non-coding RNAs in normal liver function, disease, and malignancy Long non-coding RNAs (lncRNAs) are important biological mediators that regulate numerous cellular processes. New experimental evidence suggests that lncRNAs play essential roles in liver development, normal liver physiology, fibrosis, and malignancy, including hepatocellular carcinoma and cholangiocarcinoma. In this review, we summarise our current understanding of the function of lncRNAs in the liver in both health and disease, as well as discuss approaches that could be used to target these non-coding transcripts for therapeutic purposes. # Introduction Seventy percent of the human genome is transcribed, yet only 2-3% of the genome encodes RNA transcripts that are translated into proteins. [bib_ref] Distinguishing protein-coding and noncoding genes in the human genome, Clamp [/bib_ref] Most biomedical research has focused on protein-coding genes, because proteins are considered the primary molecular building blocks that control the structure and function of cells. Recent advances in genome-wide RNA sequencing approaches have revealed the abundance and diversity of noncoding (nc) RNAs, and these RNA transcripts are increasingly recognised for their diverse biological functions in eukaryotic cells. NcRNAs are primarily classified by their mechanism of transcription and mode of action. Most ncRNAs, including ribosomal and transfer RNA molecules, are derived through transcription by RNA polymerase (RNA Pol) I and RNA Pol III. RNA Pol II also transcribes various classes of small ncRNAs including microRNAs (miRNAs), small nucleolar RNAs, and piwi-interacting RNAs, which have been reviewed in detail previously. [bib_ref] Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs, Matera [/bib_ref] The functions of longer RNA Pol II-transcribed ncRNAs, commonly referred to as long non-coding RNAs (lncRNAs) have more recently piqued the interest of researchers. [bib_ref] Large-scale transcriptional activity in chromosomes 21 and 22, Kapranov [/bib_ref] What constitutes an lncRNA is still a matter of debate. However, lncRNAs are commonly defined as ncRNA transcripts that are greater than 200 nucleotides in length, which separates them from small ncRNAs such as miRNAs. Similar to messenger (m) RNAs, lncRNAs are 5 0 -capped and commonly contain a poly-adenylated tail at their 3 0 -end. [bib_ref] lincRNAs: genomics, evolution, and mechanisms, Ulitsky [/bib_ref] Interestingly, a few lncRNAs have been described that contain an RNase P-catalysed triple-helical structure at their 3 0 -ends instead of a poly-adenylated tail, presumably increasing transcript stability. [bib_ref] Formation of triple-helical structures by the 3 0 -end sequences of MALAT1..., Brown [/bib_ref] In comparison to mRNAs, lncRNAs tend to be shorter transcripts and contain fewer exons. [bib_ref] The GENCODE v7 catalog of human long noncoding RNAs: analysis of their..., Derrien [/bib_ref] The half-lives of lncRNAs are variable, but overall lncRNAs tend to be less stable than mRNAs. [bib_ref] Genome-wide analysis of long noncoding RNA stability, Clark [/bib_ref] [bib_ref] Genome-wide analysis of long noncoding RNA turnover, Tani [/bib_ref] Decreased stability of lncRNAs helps to explain why transcription at genes encoding lncRNAs is closer in level to transcription at protein-coding genes, while there is a greater difference in expression of mature mRNAs and lncRNAs, as measured by RNA sequencing. [bib_ref] Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells, Sigova [/bib_ref] Many lncRNAs include one or multiple open reading frame (ORF) regions for protein synthesis. [bib_ref] Differentiating proteincoding and noncoding RNA: challenges and ambiguities, Dinger [/bib_ref] Yet, possible ORFs in lncRNA transcripts are often not translated or, if translated, the resulting protein product is unstable and rapidly subjected to degradation; therefore these protein products are not thought to play a substantial biological role. [bib_ref] lincRNAs: genomics, evolution, and mechanisms, Ulitsky [/bib_ref] The liver is an essential organ in the gastrointestinal system, which mediates numerous digestive and metabolic functions. Recent work from many research laboratories has led to an increased understanding of the role of lncRNAs in liver development, physiology, and pathology. We will review the identification and classification of lncRNAs, general mechanisms of action, our current understanding of lncRNA activities in the context of the liver, and the potential of lncRNAs as therapeutic targets in the treatment of liver disease. ## Identification Early efforts to identify and characterise lncRNAs employed sequencing of expressed cDNA libraries and expression sequence tags (ESTs). [bib_ref] The transcriptional landscape of the mammalian genome, Consortium [/bib_ref] However, because of the inherent laboriousness of this strategy, few lncRNAs were characterised. Subsequent approaches such as chromatin profiling by immunoprecipitation followed by high-throughput sequencing drastically improved the lncRNA discovery process. Regions containing specific histone marks, such as trimethylation of histone 3 lyisine 4 (H3K4me3) and histone 3 lysine 36 (H3K36me3), define areas of active transcriptional initiation and elongation and were employed to identify lncRNA transcripts that do not overlap with protein-coding genes. [bib_ref] Chromatin signature reveals over a thousand highly conserved large noncoding RNAs in..., Guttman [/bib_ref] With the advent of next-generation RNA-sequencing, lncRNA identification underwent a significant revolution. [bib_ref] Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching..., Trapnell [/bib_ref] With this approach, short RNA-sequencing reads were used to assemble a transcriptome model by mapping reads to a reference genome to identify exons and splice junctions between exons. The assembled transcriptome was then further subjected to a multitude of algorithms to filter out transcripts that have coding potential. [bib_ref] Methods for distinguishing between protein-coding and long noncoding RNAs and the elusive..., Housman [/bib_ref] This computational strategy involved significantly less manual work than earlier approaches. However, this method relies heavily on the error-prone de novo construction of transcript sequences from short reads, which can lead to the assembly of incomplete transcripts. With this limitation in mind, defining the 5 0 and 3 0 ends of the lncRNA through approaches such as rapid amplification of cDNA ends (RACE) [bib_ref] Rapid production of full-length cDNAs from rare transcripts: amplification using a single..., Frohman [/bib_ref] and cloning the full-length lncRNA transcript in a specific cell type of interest are good starting points to define the exons of poorly characterised lncRNAs. An alternative method that employs long-read sequencing is increasingly being utilised to sequence full-length transcripts. [bib_ref] High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing, Lagarde [/bib_ref] The promise of this newer method is the increased accuracy of the exons and splice junctions contained in each transcript, but this comes at a cost of an increased rate of sequencing errors and decreased genome coverage when performed at the same price point as short-read sequencing. With notable exceptions, most lncRNAs do not possess crossspecies sequence homology. [bib_ref] Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution, Ulitsky [/bib_ref] However, this does not by any means suggest that lncRNAs are not or cannot be evolutionarily conserved. In contrast to the conservation of protein-coding genes, which rely heavily on the sequence homology to maintain amino acid sequence, lncRNA conservation may be enforced at the level of the RNA structure, function, and or syntenic expression pattern. The last criterion, which relies on conserved location in the genome relative to proximal genes, is often used to identify human orthologs of lncRNAs that are initially found in model organisms (e.g. mice). [bib_ref] The four dimensions of noncoding RNA conservation, Diederichs [/bib_ref] Even taking these additional criteria into account, it is estimated that only a little over onethird of human lncRNAs have orthologous transcripts in mice. [bib_ref] Evolutionary dynamics and tissue specificity of human long noncoding RNAs in six..., Washietl [/bib_ref] Although these multiple levels need to be considered when addressing lncRNA conservation, it offers a far better insight into the mechanisms of lncRNA evolution across different organisms. Through these efforts, a large collection of annotated lncRNAs have been described in an increasing number of organisms and are accessible through a multitude of sources, including NON-CODE, 20 GENCODE, [bib_ref] GENCODE: the reference human genome annotation for the ENCODE Project, Harrow [/bib_ref] RNAcentral, [bib_ref] A hub of information for non-coding RNA sequences, Rnacentral [/bib_ref] and LNCipedia 23 databases. shows the number of genes encoding lncRNAs and the number of different lncRNA transcripts annotated in different species. The increased number of lncRNAs in humans and mice compared to other vertebrates likely reflects the greater depth of sequencing analysis in these two species. ## Diverse genomic origins of lncrnas LncRNAs are classified according to their sites of transcription relative to annotated protein-coding genes [bib_ref] A guide to naming human non-coding RNA genes, Seal [/bib_ref]. Transcripts that do not overlap with known protein-coding or small RNA-coding genes are termed long intervening non-coding (linc) RNAs. [bib_ref] Chromatin signature reveals over a thousand highly conserved large noncoding RNAs in..., Guttman [/bib_ref] [bib_ref] Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution, Ulitsky [/bib_ref] LincRNAs are also often described as intergenic noncoding RNAs in the literature. This category of lncRNAs was easier to identify because of their distance from known genes, and many of the earliest described lncRNAs, including H19, 25 X-inactive specific transcript (XIST), [bib_ref] A gene from the region of the human X inactivation centre is..., Brown [/bib_ref] [bib_ref] Conservation of position and exclusive expression of mouse Xist from the inactive..., Brockdorff [/bib_ref] and HOX transcript antisense RNA (HOTAIR), [bib_ref] Functional demarcation of active and silent chromatin domains in human HOX loci..., Rinn [/bib_ref] fall into this category. Natural antisense transcripts (NATs) are widely expressed in the human genome. NATs are the result of transcription on the opposite strand to the sense protein-coding gene such that the non-coding transcript is the reverse complement of a region of the protein-coding transcript. [bib_ref] The antisense transcriptomes of human cells, He [/bib_ref] NATs are understood to function predominantly in cis (i.e. acting in proximity to where they are transcribed) by regulating the sense protein-coding transcript. [bib_ref] The functions of natural antisense transcripts, Wight [/bib_ref] One mechanism proposed to explain how NATs regulate gene expression is through the formation of duplexes in which the sequence complementarity between NATs and the sense transcripts interferes with the recruitment of the splicing machinery, resulting in skipped exons or incomplete splicing through a process called RNA masking. [bib_ref] Antisense transcription: a critical look in both directions, Beiter [/bib_ref] In an alternative mechanism, the RNA duplex may serve as an immediate substrate for either RNA editing [bib_ref] Is there any sense in antisense editing?, Neeman [/bib_ref] Divergent lncRNAs are transcribed head-to-head from protein-coding genes so that the transcription start sites of the lncRNA and protein-coding genes are in close proximity and are oriented in opposite directions. Transcription of the divergent lncRNA can be facilitated by a shared bi-directional promoter that induces the expression of both lncRNA and protein-coding genes. [bib_ref] Human gene promoters are intrinsically bidirectional, Andersson [/bib_ref] A criterion that is increasingly being used to define this class of lncRNAs is whether the lncRNA transcription starts within 300-500 base pairs of the transcription start site of a protein-coding gene. [bib_ref] A guide to naming human non-coding RNA genes, Seal [/bib_ref] Enhancer RNAs (eRNA) are another novel class of RNA species and are exclusively expressed at enhancer regions. [bib_ref] Widespread transcription at neuronal activity-regulated enhancers, Kim [/bib_ref] Most eRNA transcripts are divergently transcribed (2D-eRNA) from enhancers, are composed of single exons, and are non-polyadenylated. [bib_ref] Noncoding transcription at enhancers: general principles and functional models, Natoli [/bib_ref] Because of the relatively open chromatin structure at enhancers, these eRNAs are considered to be the indirect effect of noisy transcriptional activity mediated by Pol II-containing transcriptional machinery as enhancers loop into proximity with promoters. [bib_ref] Widespread transcription at neuronal activity-regulated enhancers, Kim [/bib_ref] This transcriptional activity is primarily associated with divergently transcribed eRNAs that are not polyadenylated and has been exploited to identify novel enhancer regions by transcriptional profiling of eRNAs. [bib_ref] Discovery of active enhancers through bidirectional expression of short transcripts, Melgar [/bib_ref] Whether or not these divergent, non-polyadenylated eRNA transcripts have functional activity has been the subject of multiple studies. [bib_ref] MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription..., Zhao [/bib_ref] [bib_ref] RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation, Rahnamoun [/bib_ref] [bib_ref] Individual long non-coding RNAs have no overt functions in zebrafish embryogenesis, viability..., Goudarzi [/bib_ref] However, it is also imaginable that the process of eRNA transcription at the enhancer region could regulate enhancer activity by itself, through recruitment of activating transcription factors. [bib_ref] Noncoding transcription at enhancers: general principles and functional models, Natoli [/bib_ref] A less abundant class of eRNAs is unidirectional and polyadenylated (1D-eRNA). [bib_ref] Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters, Koch [/bib_ref] These unidirectional eRNAs have similar features to lncRNAs but are encoded by genomic regions associated with an increased abundance of H3K4me1 relative to H3K4me3 when compared to regions encoding lncRNAs. The functional distinction between a non-coding transcript labelled as a unidirectional eRNA and one labelled as an lncRNA is still not well understood, and likely represents different RNA species across the same continuum. ## Mechanisms of action: transcript or transcription, that is the question Perhaps the most studied question at the core of lncRNA biology is whether the non-coding transcript or the act of its transcription confers function to the lncRNA locus. In this line, evidence for each scenario is rapidly mounting for individual lncRNAs. For some lncRNAs, genetic manipulation of the lncRNA locus in mouse models suggests that the mere act of transcription or related processes that include splicing allow cross-regulation of lncRNAs with that of nearby protein-coding genes. [bib_ref] Local regulation of gene expression by lncRNA promoters, transcription and splicing, Engreitz [/bib_ref] However, the use of genetically manipulated models to study lncRNA function should be carefully interpreted, as complete or partial deletion of regulatory regions (i.e. promoters or enhancers) that control transcription of lncRNAs may adversely affect the expression of nearby genes. In contrast, other studies point to the direct role of transcripts, often exerted in trans, as opposed to the act of transcription, as described below. We will briefly review the range of functions attributed to lncRNA transcripts before discussing how specific lncRNAs function in the liver. ## Interactions between lncrnas and chromatin Perhaps the earliest and the best-known lncRNA that directly interacts with chromosomes to exert its function is XIST. [bib_ref] The human XIST gene: analysis of a 17 kb inactive X-specific RNA..., Brown [/bib_ref] One X chromosome is transcriptionally silenced in somatic cells of female mammals early in development in a process known as X-chromosome inactivation. [bib_ref] X-chromosome inactivation: a crossroads between chromosome architecture and gene regulation, Galupa [/bib_ref] X-chromosome inactivation begins with the expression of the XIST transcript and distribution of the transcript along the entire X chromosome. XIST assists the formation of silent heterochromatin through the recruitment of two polycomb repressive complexes (PRCs) known as PRC1 and PRC2. [bib_ref] Xist deletional analysis reveals an interdependency between xist RNA and polycomb complexes..., Colognori [/bib_ref] A host of lncRNAs have also been described that function as more targeted activators or suppressors of gene expression through modification of chromatin or DNA. HOTTIP (HOXA transcript at the distal tip), for instance, functions through the adaptor protein WDR5 to recruit histone methyltransferases to trimethylate the fourth lysine residue of histone 3 protein (H3K4me3), promoting gene expression. [bib_ref] A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression, Wang [/bib_ref] In contrast, immunoprecipitation of the PRC2 silencing complex provided the first indications that lncRNAs can direct regulatory complexes to specific loci in the genome to repress gene expression. [bib_ref] Functional demarcation of active and silent chromatin domains in human HOX loci..., Rinn [/bib_ref] HOTAIR is expressed within the HOXC cluster [bib_ref] lncRNAs: linking RNA to chromatin, Rinn [/bib_ref] [bib_ref] Linking RNA biology to lncRNAs, Goff [/bib_ref] and functions in trans (i.e. function at distant sites from transcription) to silence the HOXD loci, which is located on a different chromosome. [bib_ref] Functional demarcation of active and silent chromatin domains in human HOX loci..., Rinn [/bib_ref] Other lncRNAs such as KCNQ1OT1 recruit DNA methyltransferases in cis to catalyse CpG dinucleotide methylation and suppress gene expression in the KCNQ1 gene cluster of the paternal chromosome. [bib_ref] Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1, Mohammad [/bib_ref] Structural lncRNAs NEAT1 (nuclear enriched abundant transcript 1) was first described as an abundant nuclear lncRNA. [bib_ref] Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem..., Chen [/bib_ref] Together, with other ncRNAs, 51 NEAT1 plays a structural role to promote the formation of membrane-less nuclear bodies named paraspeckles. [bib_ref] An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential..., Clemson [/bib_ref] Paraspeckles contain both proteins and RNAs and are thought to LncRNAs are classified by genomic origins relative to protein-coding genes. Divergent lncRNAs are encoded on the opposite strand and direction from protein-coding genes. As the name suggests, lincRNAs are found in regions between genes. NATs are transcribed from the antisense strand of a protein-coding gene. 1D-eRNAs are lncRNAs transcribed from regions identified as enhancers and are distinct from 2D-eRNAs, which are divergently transcribed and nonpolyadenylated transcripts produced from enhancers. eRNA, enhancer RNA; lincRNA, long intervening non-coding RNA; lncRNA, long non-coding RNA; NATs, natural antisense transcripts; TSS, transcription start site. regulate gene expression through the retention of mRNAs in the nucleus, as recently reviewed. [bib_ref] An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential..., Clemson [/bib_ref] [bib_ref] Paraspeckles: nuclear bodies built on long noncoding RNA, Bond [/bib_ref] Similarly, another structural lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is associated with nuclear speckles, [bib_ref] The nuclearretained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing..., Tripathi [/bib_ref] which contain splicing factors. [bib_ref] Nuclear speckles: molecular organization, biological function and role in disease, Galganski [/bib_ref] Both NEAT1 and MALAT1 are present at hundreds of transcriptionally active regions and act as structural components of these two nuclear bodies. [bib_ref] The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites, West [/bib_ref] Unexpectedly, a crosstalk between NEAT1 and mitochondria was recently described. [bib_ref] Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria, Wang [/bib_ref] In response to mitochondrial stress, the transcription factor ATF2 is activated, inducing expression of NEAT1, leading to a change in nuclear paraspeckle morphology and an increase in the number of paraspeckles that retain mitochondrial mRNAs. This response results in decreased production of mitochondria and mitochondria-dependent apoptosis proteins. Thus, NEAT1 affects mitochondrial function and dynamics through changes in the expression of mitochondrial proteins. ## Lncrnas as mirna sponges One mechanism by which lncRNAs can regulate gene expression post-transcriptionally is to serve as miRNA sponges. [bib_ref] MicroRNA sponges: progress and possibilities, Ebert [/bib_ref] Because this class of lncRNAs competes with mRNAs for miRNA binding, these lncRNAs also belong to the category of competing endogenous RNAs (ceRNAs) and have been shown to regulate a vast number of genes and related pathways. [bib_ref] Competitive endogenous RNA is an intrinsic component of EMT regulatory circuits and..., Liu [/bib_ref] However, for an lncRNA to function as a miRNA sponge, it must be expressed at a high enough level to compete with mRNAs that contain the same miRNA seed sequence. LncRNAs are usually expressed at lower levels than mRNAs, and it is not clear how the stoichiometry could support this competing function for many examples of ceRNAs, even if an lncRNA contains multiple seed sequences for the same miRNA. [bib_ref] Interactions between short and long noncoding RNAs, Ulitsky [/bib_ref] ## Lncrnas in early liver development Organ development and cell fate determination are driven by the coordinated regulation of hundreds to thousands of genes. [bib_ref] Gene expression across mammalian organ development, Cardoso-Moreira [/bib_ref] Owing to their ability to modulate gene regulatory pathways and the tight control of their expression in differentiation, lncRNAs have been shown to play important roles in development. [bib_ref] Long noncoding RNAs in organogenesis: making the difference, Grote [/bib_ref] Foetal liver development is a complex process that comprises multiple differentiation stages from cells derived from different lineages.Lineage studies indicate that hepatocytes and cholangiocytes are derived from the mesendoderm lineage that in turn differentiates into the definitive endodermal (DE) layer of the early embryo. DE then organises in a tube along the anteriorposterior axis of the embryo, which ultimately forms the primitive digestive tract, including the foregut, midgut and hindgut. The foregut is the progenitor region for many internal organs including the liver, gastrointestinal system, lungs and thyroid. [bib_ref] Distinct populations of endoderm cells converge to generate the embryonic liver bud..., Tremblay [/bib_ref] Differential expression analysis of lncRNAs during DE differentiation led to the identification of the DE-specific lncRNA DEANR1 (definitive endoderm-associated lncRNA1). [bib_ref] The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression, Jiang [/bib_ref] Activin signalling drives embryonic stem cell differentiation towards DE through activation of the transforming growth factor-b (TGF-b) receptor signalling pathway and phosphorylation of the coactivators SMAD2 and SMAD3 (SMAD2/3). DEANR1 recruits SMAD2/3 to the FOXA2 promoter to induce FOXA2 expression and promote DE differentiation. Combined analysis of SMAD3 gene occupancy and expression profiling of DE differentiation led to the identification of DIGIT (divergent to goosecoid, induced by TGF-b family signalling). [bib_ref] DIGIT is a conserved long noncoding RNA that regulates GSC expression to..., Daneshvar [/bib_ref] The transcription of DIGIT, which is divergently transcribed from the gene encoding goosecoid (GSC), is induced during DE differentiation in both human and mouse models of embryonic stem cell differentiation. DIGIT interacts with BRD3 at sites of histone 3 lysine 18 acetylation (H3K18ac), regulating gene expression and promoting DE differentiation. [bib_ref] lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation, Daneshvar [/bib_ref] LncRNAs in normal liver function and lipid processing The liver is responsible for homeostasis and regulation of lipid metabolism in mammals, and disruption of normal lipid metabolism can lead to hepatic steatosis. [bib_ref] Lipid droplets and liver disease: from basic biology to clinical implications, Gluchowski [/bib_ref] Many lncRNAs have been described that affect lipid metabolism and are reviewed elsewhere. [bib_ref] Long noncoding RNAs in lipid metabolism, Van Solingen [/bib_ref] [bib_ref] Long noncoding RNAs in lipid metabolism: literature review and conservation analysis across..., Muret [/bib_ref] We will focus our discussion on lncRNAs that have been shown to regulate key steps in lipid metabolism [fig_ref] Figure 3: LncRNAs regulate various aspects of lipid metabolism [/fig_ref]. ## Lncrnas in lipid metabolism Cholesterol is imported into the liver from LDL lipoproteins via LDL receptors on hepatocytes. This influx is counterbalanced by the efflux of cholesterol through ATP-binding cassette transporter A1 (ABCA1), which delivers cholesterol to HDL lipoprotein. [bib_ref] Molecular mechanisms of cellular cholesterol efflux, Phillips [/bib_ref] Intracellular cholesterol is processed by cholesterol 7a-hydroxylase (also called cytochrome P450 family 7 subfamily A member 1 [CYP7A1]) to form 7a-hydroxycholesterol, [bib_ref] Human cholesterol 7a-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype, Pullinger [/bib_ref] which is then modified by cytochrome p450 family 8 subfamily b member 1 (CYP8B1) to eventually form cholic acid. [bib_ref] Human sterol 12a-hydroxylase (CYP8B1) is mainly expressed in hepatocytes in a homogenous..., Wang [/bib_ref] Cholic acid, in addition to chenodeoxycholic acid, is then conjugated to taurine or glycine for excretion in the bile. 74 CYP8B1 activity shifts the ratio of cholic acid:chenodeoxycholic acid in favour of cholic acid, which has a weaker stimulatory effect on the farnesoid X receptor (FXR), 75 a sensor of bile acid composition. [bib_ref] Identification of a nuclear receptor that is activated by farnesol metabolites, Forman [/bib_ref] [bib_ref] Endogenous bile acids are ligands for the nuclear receptor FXR/BAR, Wang [/bib_ref] [bib_ref] Identification of a nuclear receptor for bile acids, Makishima [/bib_ref] [bib_ref] Bile acids: natural ligands for an orphan nuclear receptor, Parks [/bib_ref] Lnc-HC was discovered as an upregulated transcript in the livers of rats with high-fat diet-induced metabolic syndrome.Although Lnc-HC is not highly evolutionarily conserved, syntenic transcripts have been found in other organisms. The study of protein interactors of this lncRNA revealed that Lnc-HC interacts with hnRNPA2B1 and forms a ribonucleoprotein complex. Analysis of data from RNA-immunoprecipitation showed that transcripts encoding CYP7A1 and ABCA1 are present in the Lnc-HC-hnRNPA2B1 complex. These two genes have previously been shown to function as key regulators of lipid and specifically cholesterol catabolism. [bib_ref] The gatekeeper for eliminating excess tissue cholesterol, Oram [/bib_ref] In vitro experiments using hepatocytes indicated that interaction with the Lnc-HC-hnRNPA2B1 complex induces nuclear retention and eventual degradation of CYP7A1 and ABCA1 transcripts, culminating in cholesterol accumulation in hepatocytes. LncLSTR (lncRNA liver-specific triglyceride regulator) is a murine lncRNA whose depletion results in reduced plasma triglyceride levels. [bib_ref] A liver-enriched long non-coding RNA, lncLSTR, regulates systemic lipid metabolism in mice, Li [/bib_ref] LncLSTR interacts with TDP-43 and interferes with the ability of TDP-43 to suppress the expression of CYP8B1, which is an essential enzyme in cholesterol metabolism. Thus, depletion of lncLSTR leads to decreased expression of CYP8B1, which affects bile acid composition. [bib_ref] Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat..., Pandak [/bib_ref] [bib_ref] Cholic acid mediates negative feedback regulation of bile acid synthesis in mice, Li-Hawkins [/bib_ref] Activation of apolipoprotein C2 (APOC2) and the reduction in plasma triglyceride levels observed with depletion of lncLSTR were attenuated with depletion of FXR. [bib_ref] Identification of a nuclear receptor that is activated by farnesol metabolites, Forman [/bib_ref] [bib_ref] Endogenous bile acids are ligands for the nuclear receptor FXR/BAR, Wang [/bib_ref] [bib_ref] Identification of a nuclear receptor for bile acids, Makishima [/bib_ref] [bib_ref] Bile acids: natural ligands for an orphan nuclear receptor, Parks [/bib_ref] Together these results suggest that lncLSTR modulates bile acid composition to regulate APOC2 expression, via FXR, [bib_ref] Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma..., Kast [/bib_ref] and to control serum triglyceride levels. Steroid response binding proteins (SREBPs) are a family of transcription factors that bind sterol response elements (SREs) in promoters to regulate the expression of genes involved in lipid metabolism. [bib_ref] SREBP-regulated lipid metabolism: convergent physiology -divergent pathophysiology, Shimano [/bib_ref] SREBPs are found as transmembrane proteins in the endoplasmic reticulum and Golgi; protein cleavage releases the DNA-binding domain which then acts as a transcription factor. [bib_ref] SREBP-1c transcription factor and lipid homeostasis: clinical perspective, Ferré [/bib_ref] In the past few years, several lncRNAs that modulate the activity of SREBPs have been characterised. Overexpression of MALAT1 in HepG2 cells, for example, increases the stability of the mRNA encoding SREBP-1c, resulting in increased intracellular lipid droplets. [bib_ref] Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing..., Yan [/bib_ref] siRNA-mediated knockdown of MALAT1 in the ob/ob mouse model of obesity results in a decrease in hepatic lipid content. These experiments also revealed that depletion of MALAT1 in the ob/ob mouse results in improved insulin sensitivity and glucose tolerance. In a similar mechanism, H19through interaction with PTBP1stabilises the SREBP-1c transcript and may also promote nuclear localisation of the SREBP-1c protein. [bib_ref] Long noncoding RNA H19 interacts with polypyrimidine tract-binding protein 1 to reprogram..., Liu [/bib_ref] Both H19 and its binding protein are increased by fatty acids in hepatocytes and mouse fatty liver models. Data also suggest that H19 is induced in patients with type 2 diabetes. [bib_ref] Epigenetic alterations in human liver from subjects with type 2 diabetes in..., Nilsson [/bib_ref] Interestingly, H19 is increased in mouse models of diabetes and temporary fasting. [bib_ref] Elevated hepatic expression of H19 long noncoding RNA contributes to diabetic hyperglycemia, Zhang [/bib_ref] Mechanistically, it is proposed that H19 also interacts with S-adenosylhomocysteine hydrolase to suppress DNA methylation 92 of the gene encoding hepatocyte nuclear factor-4a (HNF4a), resulting in increased expression of this transcriptional regulator of gluconeogenesis. Liver X receptors (LXRs) are key transcription factors that control cholesterol homeostasis. [bib_ref] Transcriptional and posttranscriptional control of cholesterol homeostasis by liver X receptors, Tontonoz [/bib_ref] Activation of LXRs results in decreased cholesterol content in the liver. [bib_ref] Liver LXRa expression is crucial for whole body cholesterol homeostasis and reverse..., Zhang [/bib_ref] LeXis (liverexpressed LXR-induced sequence) was originally characterised in cells treated with the LXR agonist GW3965. [bib_ref] Feedback modulation of cholesterol metabolism by the lipid-responsive noncoding RNA LeXis, Sallam [/bib_ref] Detailed analysis of the LeXis interactome suggests that LeXis regulates cholesterol anabolic pathways through interaction with the transcriptional coactivator RALY. In adenovirus-transduced mouse models, the induction of LeXis is associated with reduced serum cholesterol levels. LncRNAs that modulate the transfer of lipid molecules Lipoproteins are a class of lipid-binding proteins that are involved in transfer of lipid molecules in the body. Apolipoprotein A1 (APOA1) is synthesised in both the liver and intestine and is the primary protein found in HDL complexes, [bib_ref] Distribution of high density lipoprotein particles with different apoprotein composition: particles with..., Cheung [/bib_ref] which are responsible for the transport of excess cholesterol to the liver (as previously reviewed [bib_ref] Reverse cholesterol transport: high-density lipoprotein's magnificent mile, Toth [/bib_ref]. Apolipoprotein A4 (APOA4), is produced in the intestinal epithelium upon lipid absorption and is involved in a number of different physiological functions including lipid absorption, metabolism, and platelet aggregation. [bib_ref] Apolipoprotein A-IV: a multifunctional protein involved in protection against atherosclerosis and diabetes, Qu [/bib_ref] APOA1-AS is a transcript antisense to the gene encoding APOA1, which negatively regulates APOA1 transcription in both in vivo and in vitro liver models. [bib_ref] Regulation of the apolipoprotein gene cluster by a long noncoding RNA, Halley [/bib_ref] Although not much is known about the functional mechanisms of this lncRNA, it is proposed that APOA1-AS recruits various histone-modifying enzymes to the APOA1 locus, which in turn regulate APOA1 expression. Similar to APOA1-AS, APOA4-AS belongs to the class of NAT lncRNAs. [bib_ref] A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in..., Qin [/bib_ref] Expression of APOA4-AS is elevated in fatty liver disease, and depletion of this lncRNA in the ob/ob mouse model results in reduced expression of APOA4 and decreased serum triglyceride levels. APOA4-AS interacts with an RNA-stabilising protein named human antigen R (HuR) in a sequencedependent manner. The depletion of HuR protein is associated with reduced APOA4-AS and APOA4 transcript levels and suggests that interaction with HuR may help promote stability of both APOA4-AS and APOA4 mRNA. ## Lncrnas in liver injury and fibrosis Microarray profiling of lncRNAs in the whole mouse liver uncovered multiple transcripts whose expression changed significantly as a result of liver injury induced by 3 weeks of carbon tetrachloride (CCl 4 ) treatment. [bib_ref] Long non-coding RNA Gm2199 rescues liver injury and promotes hepatocyte proliferation through..., Gao [/bib_ref] Among these, Gm2199 was shown to be repressed with CCl 4 -induced damage. Treatment of hepatocytes with CCl 4 caused a significant reduction in proliferation, which was restored by ectopic expression of Gm2199. As for the mechanism, the authors provide evidence that Gm2199 achieves these effects through increased expression and activation of mitogen-activated protein kinase ERK1/2. In vivo studies corroborated these results by showing that increased expression of Gm2199 protects cells from the adverse effects of CCl 4 treatment. Although a human lncRNA orthologous to Gm2199 has not yet been described and thus Gm2199 may not have relevance for human disease, this study demonstrates how an lncRNA can affect intricate molecular pathways involved in liver injury. The list of lncRNAs identified that modulate fibrosis, in general, continues to grow and was recently reviewed elsewhere. [bib_ref] Critical effects of long non-coding RNA on fibrosis diseases, Zhang [/bib_ref] We will focus here on lncRNAs most relevant to liver fibrosis . Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrosis, [bib_ref] Hepatic lipocytes: the principal collagen-producing cells of normal rat liver, Friedman [/bib_ref] [bib_ref] Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial..., Maher [/bib_ref] [bib_ref] Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis..., Mederacke [/bib_ref] and lncRNAs expressed in human HSCs were recently described, many of which are coexpressed with extracellular matrix genes. [bib_ref] Long noncoding RNAs expressed in human hepatic stellate cells form networks with..., Zhou [/bib_ref] In mouse models of CCl 4 -induced liver fibrosis, lincRNA-p21 was found to be significantly decreased. Overexpression of lincRNA-p21 in primary HSCs resulted in increased expression of the p21 protein and reduced expression of a-smooth muscle actin (ɑ-SMA [ACTA2]) and collagen type I a 1 (COL1A1), suggesting a role for lncRNA-p21 in suppressing HSC activation and transformation to myofibroblasts. [bib_ref] lincRNA-p21 inhibits hepatic stellate cell activation and liver fibrogenesis via p21, Zheng [/bib_ref] MEG3 (maternally expressed gene 3) is a maternally imprinted gene whose non-coding product has been described in multiple cancers. 108,109 MEG3 expression is decreased in fibrotic liver tissue and activated in primary HSCs. Overexpression of this lncRNA through a viral induction system also showed antifibrotic activity in murine CCl 4 -induced liver fibrosis. [bib_ref] LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212, Yu [/bib_ref] [bib_ref] Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation..., He [/bib_ref] In the immortalised human HSC line LX-2, MEG3 transcript levels are reduced in response to TGF-b treatment. [bib_ref] Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation..., He [/bib_ref] The mechanism by which MEG3 confers antifibrotic activity is still largely unknown. ## Review MEG3 is suggested to inhibit HSC activation by suppressing epithelial-mesenchymal transition through interactions with smoothened (SMO) protein. MEG3 has also been shown to induce cholestatic liver damage by accelerating the decay of small heterodimer partner (SHP) mRNA through interactions with PTBP1. [bib_ref] Long noncoding RNA MEG3 induces cholestatic liver injury by interaction with PTBP1..., Zhang [/bib_ref] In turn, attenuation of SHP protein, which is a key repressor in the bile acid biosynthesis pathway, results in liver injury. The role of GAS5 (growth arrest-specific transcript 5) in liver fibrosis has also been described. [bib_ref] Long non-coding RNA growth arrest-specific transcript 5 (GAS5) inhibits liver fibrogenesis through..., Yu [/bib_ref] Expression of GAS5 is decreased in human and mouse fibrotic liver tissues. Similarly, in vitro experiments employing activated HSCs also suggest that GAS5 expression is reduced compared to quiescent HSCs. Overexpression of this lncRNA results in decreased expression of COL1A1 and ACTA2 in HSCs in vitro and decreased collagen levels in CCl 4 -induced murine liver fibrosis models, as determined by measurement of hepatic hydroxyproline content. Mechanistically, GAS5 is considered to function as a ceRNA through interaction with miR-233. The authors demonstrated that sequestration of miR-233 by GAS5 results in increased expression of the miRNA target transcripts including the p27 gene, whose protein product is directly involved in HSC proliferation and activation. H19 is induced in cholestatic liver fibrosis. [bib_ref] H19 promotes cholestatic liver fibrosis by preventing ZEB1-mediated inhibition of epithelial cell..., Song [/bib_ref] Ectopic expression of H19 in the liver is associated with increased necrosis and fibrosis in the setting of bile duct ligation (BDL), and H19-deficient mice show reduced cholestatic liver injury and fibrosis in response to BDL. H19 exerts its function in this setting by binding and inhibiting the zinc finger E-box-binding homeobox 1 (ZEB1), whose activity suppresses activation of epithelial cell adhesion molecule (EpCAM). Ectopic expression of ZEB1 or depletion of EpCAM in H19-deficient mice reduced fibrosis, leading to a model where bile acids induce H19 expression in hepatocytes and cholangiocytes. H19 then interacts with ZEB1, preventing binding to the EpCAM promoter, leading to increased EpCAM expression and increased susceptibility to cholestatic liver fibrosis. ## Lncrnas in liver cancer LncRNAs are increasingly recognised as mediators of human cancers. [bib_ref] Long noncoding RNAs in cancer pathways, Schmitt [/bib_ref] In this regard, depending on the context, lncRNAs can function as either oncogenes or tumour suppressors. In the liver, lncRNAs have been identified that are associated with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) [fig_ref] Table 1: LncRNAs associated with liver cancer [/fig_ref] , which comprise the majority of liver cancer cases, and were reviewed recently. [bib_ref] Roles and regulation of long noncoding RNAs in hepatocellular carcinoma, Lim [/bib_ref] Below, we describe oncogenic lncRNAs that are involved in promoting HCC and CCA; we then look at lncRNAs that function as tumour suppressors to hinder malignant growth. ## Oncogenic lncrnas As the name suggests, MALAT1, also known as NEAT2 (nuclearenriched abundant transcript 2), is an oncogenic lncRNA in human cancer. [bib_ref] MALAT-1, a novel noncoding RNA, and thymosin b4 predict metastasis and survival..., Ji [/bib_ref] This lncRNA is highly conserved in mammals, and its role in numerous biological processes has been extensively studied. In HCC, MALAT1 is upregulated, and its depletion results in lower tumorigenicity, which is largely driven by reduced expression of the oncogenic splicing factor SRSF1. [bib_ref] Long noncoding RNA MALAT1 promotes hepatocellular carcinoma development by SRSF1 upregulation and..., Malakar [/bib_ref] Furthermore, MALAT1 activates the mammalian target of rapamycin (mTOR) pathway through SRSF1-mediated splicing of S6 kinase 1 (S6K1) to an alternative isoform named Iso-2. The Iso-2 protein product has oncogenic potential through direct binding and activation of mTOR complex 1 (mTORC1). We discussed the role of NEAT1 as a structural lncRNA previously. However, NEAT1 has also been implicated in multiple cancers, [bib_ref] Long noncoding RNA NEAT1 confers oncogenic role in triple-negative breast cancer through..., Shin [/bib_ref] [bib_ref] Oncogenic properties of NEAT1 in prostate cancer cells depend on the CDC5L-AGRN..., Li [/bib_ref] and its role and expression status in HCC have recently been described. [bib_ref] Long non-coding RNA NEAT1 promotes hepatocellular carcinoma cell proliferation through the regulation..., Fang [/bib_ref] Expression profiling of NEAT1 transcripts in normal liver and HCC tumour tissues and cell lines revealed that NEAT1 is induced in neoplastic cells. Fang et al. ## Gas5 Tumour suppressor Functions as a sponge to bind miRNAs. Binds to miR-126-3p, whose ectopic expression has been shown to decrease cell proliferation in HCC. Binds to miR-21 to derepress the PTEN protein levels [bib_ref] Differential expression profiling of long non-coding RNA GAS5 and miR-126-3p in human..., Faranda [/bib_ref] Cholangiocarcinoma (CCA) DANCR/ANCR Oncogene Associated with increased cellular proliferation and migration through interaction with EZH2 [bib_ref] Long noncoding RNA DANCR regulates proliferation and migration by epigenetically silencing FBP1..., Wang [/bib_ref] In HCC, lncRNAs have been described to function as both oncogenes and tumour suppressors. In CCA, DANCR/ANCR has been shown to function as an oncogene. suggested that, mechanistically, NEAT1 exerts its oncogenic property as a ceRNA through binding and inhibition of miR-129-5p, whose main cellular targets include valosin-containing protein (VCP) and the NF-jB inhibitor protein (IjB). HEIH (high expression in HCC) was originally identified in HBV-associated HCC tissues. [bib_ref] Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through..., Yang [/bib_ref] RNA pulldown assays indicated that HEIH interacted with the PRC2 complex protein EZH2. shRNA-mediated depletion of HEIH caused increased expression of genes that are normally repressed by the PRC2 complex. HEIH promotes cell cycle progression and tumour growth via a pathway that is at least in part dependent on EZH2. This study also found that the expression level of HEIH in tumour tissue has potential as a prognostic marker in patients with HCC HULC (highly upregulated in liver cancer) is an oncogenic lncRNA whose role, among others, has been described in hepatic, gastric, and colorectal cancers. [bib_ref] Characterization of HULC, a novel gene with striking up-regulation in hepatocellular carcinoma,..., Panzitt [/bib_ref] Abnormal expression of HULC is associated with poor prognosis of pancreatic cancers and hepatomas in the clinic. [bib_ref] Long noncoding RNA HULC is a novel biomarker of poor prognosis in..., Peng [/bib_ref] [bib_ref] Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through..., Cui [/bib_ref] Measuring serum levels of this lncRNA has also been proposed as a biomarker in liver cancer. [bib_ref] Plasma HULC as a promising novel biomarker for the detection of hepatocellular..., Xie [/bib_ref] [bib_ref] HULC and Linc00152 act as novel biomarkers in predicting diagnosis of hepatocellular..., Li [/bib_ref] HULC influences many cell traits in cancer through multi-pronged metabolic and cellular signalling pathways that are all relevant in hepatic biology. The transcription factor CREB and the HBV X protein (HBx) have been well-studied in the context of liver cancer [bib_ref] HULC: an oncogenic long noncoding RNA in human cancer, Yu [/bib_ref] and are upstream regulators of HULC. HULC can act as a miRNA sponge. It contains binding sites for miR-372, which usually targets translation of the CREB-kinase PRKACB. PRKACB-mediated phosphorylation of CREB results in chromatin modifications that promote gene expression including that of HULC. [bib_ref] CREB up-regulates long noncoding RNA, HULC expression through interaction with microRNA-372 in..., Wang [/bib_ref] Additionally, infection with HBV is associated with increased levels of HULC in HCC samples. Detailed analysis of HBV-induced HCCs has demonstrated that HBV promotes the expression of HULC. [bib_ref] Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus..., Du [/bib_ref] Interestingly, HULC has also been shown to accelerate liver cancer via inhibition of the PTEN tumour suppressor gene through sponge-activity on miR-15a. [bib_ref] Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy..., Xin [/bib_ref] HULC also promotes lipogenesis in HCC. Cholesterol molecules upregulate the expression of HULC by activating the retinoic receptor RXR-ɑ. [bib_ref] Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through..., Cui [/bib_ref] Increased expression of HULC is associated with increased methylation of the gene encoding miR-9. This results in decreased expression of miR-9 and increased expression of its target, the nuclear receptor peroxisome proliferator-activated receptor-a (PPAR-ɑ). Increased levels of PPAR-ɑ then induce transcription of long-chain-fatty-acid-CoA ligase 1 (ACSL1), to promote fatty acid synthesis. HOTTIP is induced in HCC. [bib_ref] Long noncoding RNA HOTTIP/HOXA13 expression is associated with disease progression and predicts..., Quagliata [/bib_ref] [bib_ref] Long noncoding RNA HOTTIP expression predicts tumor recurrence in hepatocellular carcinoma patients..., Wu [/bib_ref] Studies have shown that the HOTTIP gene is located near to the HOXA13 gene and controls the gene expression of the HOXA loci. The HOXA locus codes for a multitude of transcription factors that are involved in embryogenesis, with aberrant expression of these factors implicated in cancers. [bib_ref] Transcriptional and posttranscriptional regulation of HOXA13 by lncRNA HOTTIP facilitates tumorigenesis and..., Lin [/bib_ref] HOTAIR was originally identified through microarray tiling of the HOXC locus on chromosome 12. [bib_ref] Functional demarcation of active and silent chromatin domains in human HOX loci..., Rinn [/bib_ref] HOTAIR repressively alters the chromatin landscape of promoters through recruitment of PRC2. In a separate but related mechanism, HOTAIR recruits the lysine-specific demethylase 1 (LSD1) complex, which is composed of LSD1, REST and CoREST proteins, and mediates demethylation of activating histone marks (H3K4me3). [bib_ref] Histone demethylation mediated by the nuclear amine oxidase homolog LSD1, Shi [/bib_ref] [bib_ref] An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation, Lee [/bib_ref] The combined action of PRC2 and LSD1 complexes suppresses target gene expression. The role of HOTAIR in HCC has been extensively studied and reviewed elsewhere. [bib_ref] Role of the long non-coding RNA HOTAIR in hepatocellular carcinoma, Wu [/bib_ref] Aberrant expression of HOTAIR is correlated with worse outcomes in patients with HCC. H19 is discussed above as a factor that regulates lipid metabolism and liver fibrosis; its role in HCC is less clear. In one study, increased expression of H19 was identified in the setting of hypoxia and metastatic disease, suggesting possible oncogenic activity, [bib_ref] The H19 non-coding RNA is essential for human tumor growth, Matouk [/bib_ref] while in HCC cell lines ectopic expression of H19 was shown to induce cell apoptosis and inhibit cell proliferation. [bib_ref] The long non-coding RNA H19 suppresses carcinogenesis and chemoresistance in hepatocellular carcinoma, Schultheiss [/bib_ref] Further data from mouse models found that expression of H19 from a modified H19 locus negatively correlates with tumour emergence in SV40-induced HCCs. [bib_ref] The H19 locus acts in vivo as a tumor suppressor, Yoshimizu [/bib_ref] DANCR (differentiation antagonising non-protein coding RNA), also known as ANCR, has been implicated in many cancers. [bib_ref] Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain..., Malakootian [/bib_ref] [bib_ref] Silencing of long-non-coding RNA ANCR suppresses the migration and invasion of osteosarcoma..., Liu [/bib_ref] DANCR is highly expressed in HCC and promotes cellular proliferation and metastasis. [bib_ref] LncRNA ANCR promotes hepatocellular carcinoma metastasis through upregulating HNRNPA1 expression, Wen [/bib_ref] In HepG2-induced xenograft mouse models of HCC, depletion of DANCR results in reduced tumour burden and decreased metastasis. The main mechanism through which DANCR exerts its oncogenic role is through interaction with the heterogenous nuclear protein ribonucleoprotein A1 (HNRNPA1), which regulates epithelialmesenchymal transition in HCC. DANCR directly binds to the HNRNPA1 protein resulting in its increased stability. DANCR has also been shown to have sponge activity, binding to miR-140-3p to inhibit its ability to affect HNRNPA1 translation. While the role of lncRNAs in liver cancers has mostly been described in HCCs, at least one lncRNA has been described in the context of CCA. CCA is an aggressive form of liver cancer whose malignant cells originate from bile ducts. [bib_ref] Cholangiocarcinoma -evolving concepts and therapeutic strategies, Rizvi [/bib_ref] DANCR is induced in CCA in addition to HCC. In vitro analysis of DANCR in CCA models shows that this lncRNA is associated with increased cell proliferation and migration through interaction with EZH2, which leads to epigenetic suppression of the fructose-bisphosphatase 1 (FBP1) gene. [bib_ref] Long noncoding RNA DANCR regulates proliferation and migration by epigenetically silencing FBP1..., Wang [/bib_ref] FBP1 is an enzyme that regulates gluconeogenesis and is implicated in multiple forms of cancer including HCCs. [bib_ref] Fructose-1, 6-bisphosphatase opposes renal carcinoma progression, Li [/bib_ref] [bib_ref] Loss of FBP1 by snail-mediated repression provides metabolic advantages in basal-like breast..., Dong [/bib_ref] [bib_ref] Decreased expression of fructose-1,6-bisphosphatase associates with glucose metabolism and tumor progression in..., Hirata [/bib_ref] While DANCR appears to function as an oncogene in both HCC and CCA, the described mechanisms of action for DANCR in these 2 liver malignancies are currently different. Future studies will be required to understand if interactions with HNRNPA1 also play a role in CCA and if interactions with EZH2 also play a role in HCC. ## Tumour suppressor lncrnas In addition to having roles in liver fibrosis as discussed before, MEG3 is also implicated in cervical, pancreatic, and many other cancers. [bib_ref] Long non-coding RNA MEG3 inhibits cervical cancer cell growth by promoting degradation..., Zhang [/bib_ref] [bib_ref] Long non-coding RNA MEG3 functions as a tumour suppressor and has prognostic..., Ma [/bib_ref] Whereas other lncRNAs appear to promote oncogenesis, MEG3 is largely thought to function as a tumour suppressor gene. [bib_ref] Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell..., Zhang [/bib_ref] [bib_ref] LncRNA MEG3 inhibits the progression of prostate cancer by facilitating H3K27 trimethylation..., Zhou [/bib_ref] Compared to normal cells, malignant hepatocytes show a significant reduction in the expression of this lncRNA. [bib_ref] microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in..., Braconi [/bib_ref] Overexpression of MEG3 in both cancer cell lines and mouse models inhibits growth of liver cancer cells. [bib_ref] Long noncoding RNA MEG3 suppresses liver cancer cells growth through inhibiting b-catenin..., Zheng [/bib_ref] The authors suggest that MEG3 increases expression of miR-122 through which pyruvate kinase muscle isozyme M2 (PKM2) is downregulated. PKM2 is an important metabolic enzyme in glycolysis that influences oncogenesis. [bib_ref] PKM2 and cancer: the function of PKM2 beyond glycolysis, Dong [/bib_ref] As a miRNA-sponge, FENDRR (foetal-lethal non-coding developmental regulatory RNA) has a unique role in HCC development. Compared to normal cells and tissues, FENDRR levels are drastically lower in HCC cell lines and tissues. [bib_ref] Long non-coding RNA FENDRR acts as a miR-423-5p sponge to suppress the..., Yu [/bib_ref] The sponge activity of FENDRR is exerted through binding to miR-423-5p, which targets and degrades growth arrest and DNAdamage-inducible beta protein gene (GADD45B) transcripts. Lower expression of the lncRNA in HCC allows the miRNA to attenuate GADD45B protein levels, which results in unchecked proliferation and immune evasion of tumour cells. Forced expression of FENDRR in mice resulted in decreased burden and proliferation of cancer cells. Progression of liver cancer is thought to be mediated through a class of stem-like cancer cells termed liver cancer stem cells (LCSCs), which are capable of sustaining proliferation within the tumour. [bib_ref] Cancer stem cells: impact, heterogeneity, and uncertainty, Magee [/bib_ref] DILC (downregulated in liver cancer stem cells) has been identified to play crucial roles in these cells. [bib_ref] Long non-coding RNA DILC regulates liver cancer stem cells via IL-6/STAT3 axis, Wang [/bib_ref] Depletion of this lncRNA resulted in expansion of the LCSC population and progression of disease, whereas ectopic expression led to a reduction in the size of the LCSC population in the tumour. Mechanistically, DILC is thought to interact with the IL6 promoter leading to its inhibition. IL6 suppression disrupts the IL6/STAT3 autocrine signalling pathway, which is required for the maintenance of LCSCs. GAS5 has been implicated in multiple forms of cancers, [bib_ref] The role of long non-coding RNA GAS5 in cancers, Ji [/bib_ref] and is a well-known tumour suppressor gene. [bib_ref] Genes specifically expressed at growth arrest of mammalian cells, Schneider [/bib_ref] However, its multitude of roles in HCC have only recently been investigated. [bib_ref] Association between indel polymorphism in the promoter region of lncRNA GAS5 and..., Tao [/bib_ref] [bib_ref] Differential expression profiling of long non-coding RNA GAS5 and miR-126-3p in human..., Faranda [/bib_ref] [bib_ref] Downregulation of LncRNA GAS5 promotes liver cancer proliferation and drug resistance by..., Wang [/bib_ref] Wang et al. showed that GAS5 expression in HCC is lower than in normal cells and tissues, and depletion of GAS5 increased resistance to the chemotherapeutic agent doxorubicin. The authors found that GAS5 acts as a sponge to inhibit oncogenic miR-21, which targets the tumour suppressor gene PTEN. In contrast, Faranda et al. also reported that the GAS5 transcript functions as a sponge to bind miR-126-3p, whose ectopic expression has been shown to decrease cell proliferation in HCC model cell lines. Although the authors did not elaborate on the mechanistic function of this miRNA in the context of HCC, studies in endothelial cells suggest that miR-126 acts through the downregulation of Spred-1, which is a negative regulator of the mitogen-activated protein kinase signalling pathway. [bib_ref] The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis, Wang [/bib_ref] Targeting lncRNAs? Studies suggest that the expression of lncRNAs is more tissue specific than that of protein-coding mRNAs. [bib_ref] High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity, Mattioli [/bib_ref] This observation makes lncRNAs attractive targets for therapeutic intervention in cases where tissue-specific modalities are highly desirable. As a major metabolic organ, the liver also has a remarkable ability to absorb external therapeutic agents. [bib_ref] Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks..., Soni [/bib_ref] [bib_ref] High levels of foreign gene expression in hepatocytes after tail vein injections..., Zhang [/bib_ref] [bib_ref] Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA, Liu [/bib_ref] Because of this property, the employment of agents to deplete lncRNAs could be an effective approach to affect the gene expression of specific cell types in the liver. The best-studied class of molecules that through direct and specific interactions affect RNA transcript levels are antisense oligonucleotides (ASOs). [bib_ref] Pharmacology of antisense drugs, Bennett [/bib_ref] ASOs constitute a large family of chemically related molecules that exert their function through different cellular mechanisms. [bib_ref] Origins and mechanisms of miRNAs and siRNAs, Carthew [/bib_ref] For example, double-stranded ASOs such as small interfering RNAs (siRNA) form a complex with Argonaute protein that actively cleaves the target transcripts in the cytoplasm, [bib_ref] MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies, Liu [/bib_ref] whereas single-stranded ASOs such as locked nucleic acids activate RNase H to cleave target transcripts in both the nucleus and cytoplasm. [bib_ref] The rates of the major steps in the molecular mechanism of RNase..., Vickers [/bib_ref] The choice between siRNA or locked nucleic acid should, therefore, consider the cellular localisation of target transcripts. Irrespective of the mechanism, cleaved RNAs are quickly degraded by various exonucleases. [bib_ref] RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by..., Lima [/bib_ref] Multiple ASOs are currently being employed clinically to target liver-based gene expression. Mipomersen, for example, targets apolipoprotein B-100, a component of LDL, to treat patients with familial hypercholesterolemia disorder. [bib_ref] Lomitapide and mipomersen: two first-in-class drugs for reducing low-density lipoprotein cholesterol in..., Rader [/bib_ref] However, the clinical use of ASOs, including mipomersen, is not without caveats and drawbacks, as ASOs may elicit an interferon response and tissue toxicity. [bib_ref] Patisiran: first global approval, Hoy [/bib_ref] Patisiran is another successful example of an ASO approved for clinical use. Patisiran, a lipid nanoparticlecontaining double-stranded RNA, is used to treat polyneuropathy of hereditary transthyretin (TTR)-mediated amyloidosis. Partisiran binds to TTR mRNA and causes its degradation resulting in a reduction in serum and tissue deposition of TTR proteins. We envision lncRNAs can be targeted using similar approaches. Conjugation of chemical groups to ASOs, as a method to increase half-life and tropism to a specific tissue or cell type, has also been examined. For example, conjugation of cholesterol molecules to ASOs enhances their absorption by hepatocytes. [bib_ref] Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs, Soutschek [/bib_ref] Hepatocytes express asialoglycoprotein receptors whose function is to clear aged circulating serum glycoproteins that have lost terminal sialic acid moieties in their glycan chain, resulting in exposure of N-acetylgalactosamine residues. [bib_ref] Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal..., Schlesinger [/bib_ref] For this reason, N-acetylgalactosamine-conjugated molecules have been under active investigation for hepatocyte-specific delivery of siRNAs. [bib_ref] GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics, Springer [/bib_ref] [bib_ref] Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense..., Tanowitz [/bib_ref] The delivery of ASOs through lipid-containing vehicles is also under active consideration. Liposome-mediated delivery of ASOs also enhances the efficiency and tropism of liver cells. Sato et al. exploited vitamin A-coupled liposome systems to introduce ASOs (targeting the collagen chaperone gp46) to rat HSCs in order to inhibit cirrhosis. [bib_ref] Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against..., Sato [/bib_ref] They found that liposome-mediated delivery of the ASO caused a marked reduction in liver fibrosis and increased survival in rat models. As an alternative ASO delivery method, lipid-like nanoparticles have also been used to introduce ASOs into the liver. [bib_ref] Specific hepatic delivery of procollagen a1(I) small interfering RNA in lipid-like nanoparticles..., Calvente [/bib_ref] Lipid-like nanoparticle-siRNAs against procollagen ɑ-I were primarily localised to nonparenchymal cells in the liver and caused a significant reduction in collagen expression and deposition of collagen in mouse liver tissue. With increased efficiency in the delivery of ASOs to specific cell types in the liver, we anticipate seeing the development of ASO-based drugs that target disease-relevant lncRNAs in the not too distant future. Although most of our discussion has focused on in vivo depletion of lncRNAs, delivery of lncRNAs to specific cell types in the liver could also have significant therapeutic value for managing chronic liver disease and cancer. The methods of RNA delivery in general and lncRNA in particular, as well as methods to induce their expression in tissues, are still in their infancy. Adeno-associated virus-based delivery methods have been acclaimed as the current gold standard for tissue delivery of nucleic acids in model organisms. [bib_ref] Adeno-associated virus vector as a platform for gene therapy delivery, Wang [/bib_ref] The rather obvious drawback of using this system is that it is largely limited by lncRNA size and concern over carcinogenicity of the virus-delivery method. [bib_ref] Long noncoding RNA facilitated gene therapy reduces atherosclerosis in a murine model..., Tontonoz [/bib_ref] Due to the unstable nature of RNAs and their large size, methods to directly deliver lncRNA molecules (as nanoparticles or other vehicles) to human tissues have not been well developed. With the development of safer and more effective delivery methods, the introduction of lncRNAs may have great potential in liver-based gene therapy. ## Lncrnas as biomarkers in diagnostic settings Because of their tissue-specific expression, lncRNAs are being investigated as biomarkers in clinical settings. [bib_ref] The role of non-coding RNAs in oncology, Slack [/bib_ref] In liver cancer, studies and meta-analyses to identify lncRNAs that could be used as biomarkers have already begun. [bib_ref] Diagnostic value of lncRNAs as biomarker in hepatocellular carcinoma: an updated meta-analysis, Chen [/bib_ref] Through analysis of data generated from the cancer genome atlas (TCGA) consortium, Li et al. constructed mRNA-lncRNA coexpression networks. The expression pattern of coding and non-coding transcripts in HCC led to the identification of several lncRNAs that have potential as biomarkers for HCC. [bib_ref] Identification of diagnostic long non-coding RNA biomarkers in patients with hepatocellular carcinoma, Li [/bib_ref] Although these lncRNAs were not able to predict patient outcomes, they hold promise in the diagnostic setting. As described earlier, perhaps the most promising lncRNA biomarker in HCC is HULC. HULC transcripts are significantly elevated in the plasma of patients with HCC. [bib_ref] Plasma HULC as a promising novel biomarker for the detection of hepatocellular..., Xie [/bib_ref] [bib_ref] HULC and Linc00152 act as novel biomarkers in predicting diagnosis of hepatocellular..., Li [/bib_ref] Additionally, in comparison to serum derived from healthy individuals, higher levels of HULC transcripts are found in the serum of patients with positive HBV status. Detection of lncRNAs such as HULC in plasma suggests that lncRNAs have the potential to serve as blood-based biomarkers in the clinical setting. # Conclusion LncRNAs play essential roles in early liver development, the metabolic function of the liver, liver fibrosis, and cancer. While lncRNAs have been identified that regulate each of these stages, these transcripts likely represent only a small fraction of the lncRNAs that control key processes from liver development to homeostasis to disease. As we gain a deeper understanding of the diversity and function of lncRNAs across the different cell types of the liver, we anticipate this knowledge will lead to the establishment of lncRNAs as therapeutic targets to treat diseases of, or regulated by, the liver; this knowledge could also be used to identify new biomarkers to track or predict disease progression. Abbreviations ABCA1, ATP-binding cassette transporter A1; ACTA2/ɑ-SMA, a-smooth muscle actin; APO, apolipoprotein; ASO, antisense oligonucleotides; BDL, bile duct ligation; CCA, cholangiocarcinoma; CCl 4 , carbon tetrachloride; ceRNA, competing endogenous RNA; COL1A1, collagen type I a 1; CYP, cytochrome P450; DE, definitive endoderm; DANCR, differentiation antagonising non-protein coding RNA; DEANR1, definitive endodermassociated lncRNA1; DIGIT, divergent to goosecoid, induced by TGF-b family signalling; DILC, downregulated in liver cancer stem cells; EpCAM, epithelial cell adhesion molecule; eRNA, enhancer RNAs; EST, expression sequence tag; FBP1, fructose-bisphosphatase 1; FENDRR, foetal-lethal non-coding developmental regulatory RNA; FXR, farnesoid X receptor; GAS5, growth arrest-specific transcript 5; H3K4me3, histone 3 lysine 4 trimethylation; H3K18ac, histone 3 lysine 18 acetylation; H3K36me3, histone 3 lysine 36 trimethylation; HCC, hepatocellular carcinoma; HEIH, high expression In HCC; HNRNPA1, heterogenous nuclear protein ribonucleoprotein A1; HOTAIR, HOX transcript antisense RNA; HOTTIP, HOXA transcript at the distal tip; HSC, hepatic stellate cells; HuR, human antigen R; HULC, highly upregulated in liver cancer; LCSC, liver cancer stem cell; LeXis, liver-expressed LXR-induced sequence; lincRNA, long intervening non-coding RNA; lncRNA, long non-coding RNA; LncLSTR, lncRNA liverspecific triglyceride regulator; LSD1, lysine-specific demethylase 1; LXR, liver X receptors; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; MEG3, maternally expressed gene 3; mTOR, mammalian target of rapamycin; NAT, natural antisense transcript; NEAT1, nuclear enriched abundant transcript 1; ORF, open reading frame; PKM2, pyruvate kinase muscle isozyme M2; PPAR-a, peroxisome proliferatoractivated receptor-a; PRC, polycomb repressive complex; RACE, rapid amplification of cDNA ends; RNA Pol, RNA polymerase; S6K1, S6 kinase 1; SHP, small heterodimer partner; siRNA, small interfering RNA; SREBPs, steroid response binding proteins; SREs, sterol response elements; TGF-b, transforming growth factor-b; TTR, transthyretin; XIST, X-inactive specific transcript; ZEB1, zinc finger E-box-binding homeobox 1. ## Financial support ACM is supported by NIH/NIDDK grant R01DK116999, NIH/NICHD grant R01HD09277302 and a Pew Biomedical Scholars Award. Dr. Mullen also receives research support from the Chan Zuckerberg Initiative and has received research funding from Boehringer Ingelheim, Bristol-Myers Squibb, Takeda Pharmaceuticals and Roche Pharmaceuticals in the last 36 months. [fig] Figure 3: LncRNAs regulate various aspects of lipid metabolism. Extrahepatic cholesterol enters hepatocytes through the LDL receptor (LDLR). lncRNAs lnc-HC and lncLSTR control key enzymes in cholesterol catabolism. H19 and MALAT1 regulate SREBP-1c stability. SREBP-1c controls the expression of genes regulating fatty acid synthesis including ACLY (ATP citrate synthase), ACC (acetyl-CoA carboxylase), FASN (fatty acid synthase) and ELOVL1 (ELOVL fatty acid elongase 1).186 HNF4a, which regulates critical genes such as PEPCK in gluconeogenesis, is inhibited by H19. Changes in the ratio of bile acids triggers FXR activation as a transcription factor to promote the expression of APOC2. Lipoproteins including APOA1 and APOA4 are involved in transfer of lipid molecules and are regulated by APOA1-AS and APOA4-AS. ER, endoplasmic reticulum. [/fig] [table] Table 1: LncRNAs associated with liver cancer. [/table]

Environmental Factors and Ventilation Affect Concentrations of Microorganisms in Hospital Wards of Southern Thailand Hospitals tend to have high density of occupancy. Poor indoor environmental quality in hospital buildings can exacerbate the health problems of patients and also harm visitors and staff. is study investigated the environmental characteristics and ventilation affecting the concentration of microorganisms in multiple-bed hospital wards. e measurements were accomplished by using a biosampler and an open plate method at four wards, different positions of electric fans, and different times. Data were analyzed by t-test and MANOVA. e results revealed that the concentrations of airborne bacteria were higher than the concentrations of fungi. ere were significant differences in the concentrations of bacteria and fungi between sampling times and between hospital wards (p < 0.05), while no difference was observed by positions of electric fans. Correlations between the concentrations and other environmental parameters indicate that temperature, number of occupants, and humidity were physical factors associated with the concentrations of microorganisms. In addition, mostly, Gram-positive bacteria were observed. is indicates the conditions in buildings in the tropical climate, and regular housekeeping of all room areas is needed to prevent the growth of airborne contaminants and the related risks to patients, visitors, and staff. # Introduction Hospitals are dynamic environments exposed to various indoor and outdoor environmental sources and can support microbial survival and growth. Characterizing the airborne microorganisms in hospital buildings is important. ere is an opportunity to spread infections via air by coughing, conversation, laughing, or sneezing. Outbreaks of infectious diseases, such as tuberculosis, measles, and influenza, have been linked with poor microbiological quality of hospital indoor air. e presence of nosocomial infectious microorganisms is a source of nosocomial infections in hospitals. ese conditions may negatively impact the health of those working in such buildings, and microbial contamination may cause diseases. Several studies have shown that microbial contamination of indoor air in a hospital is mostly by bacteria and fungi. ere is increasing evidence of high airborne concentrations of bacteria in hospitals of Singapore. e wards are potentially risky places for human occupants in a hospital. Investigation of airborne concentrations in different wards of a hospital was carried out in Isfahan, and high levels of airborne bacteria were observed. Previous studies have focused on airborne concentrations of bacteria, while the concentrations of fungi have received less attention. In ailand, Srion and Nathapindhufound bacteria on about half the sampled surfaces, in Nonsang and Nongbua Lamphu hospitals. Also, seven genera of bacteria and fungi were identified in the air of Khonkaen hospital. e environmental characteristics affecting airborne concentrations have not yet been determined. e presence of high concentrations of bacteria and fungi within hospital buildings is related to the number of occupants, their activities, and ventilation. Microorganisms that are major causes of illness had increased concentrations in the environment affecting visitors. e high concentration of bacteria or fungi in the air indicates poor ventilation. Proper air ventilation is one method that can reduce the air pollution and the airborne microorganisms in buildings. e strategy in the design of the ventilation system of the building is to control the environmental parameters, including air movement, humidity, temperature, air velocity, and cleanliness. Generally, the microbiological quality indoors was not higher than the air quality standards and not higher than the outdoors. It is important to reduce and control the concentrations of harmful microorganisms in the air, to ensure good air quality. Several studieshave indicated that an important aspect in the work environment is the efficiency of the ventilation system and suggested associations among sources, risk factors, and transmission of microorganisms in the hospital and that air cleaning could decrease the concentrations. In southern ailand, high temperature and humidity favor airborne microorganisms, and poor indoor air quality in hospitals may cause diverse healthcare-associated infections of patients and staff. Our prior indoor air quality (IAQ) studies have reported on concentrations of Aspergillus spp., Cladosporium spp., and Penicillium spp. in three schools. ey indicated that these fungi comprise a risk factor at the school, potentially inducing asthma symptoms in the students. Previous studieshave indicated that air pollution sources (e.g., volatile organic compounds (VOCs), nitrogen dioxide (NO 2 ), radon, ventilation, formaldehyde, humidity, temperature, and biocontaminants) might have negative effects on indoor air quality in schools. ese studies suggested that the importance of natural ventilation could be focused and increased for improving indoor air quality and children's health. Schools, like hospitals, as communal premises, are critical environments due to the long stay and exposure of vulnerable groups of people. us, indoor air quality represents an important issue in both hospitals and schools due to the highest risk of exposure to contaminants, which can accumulate particularly when correct air ventilation is not implemented. Prasomwong et al.sampled a hotel in ailand, and the measurement results showed that humidity and temperature were both above recommended limits, and poor air quality was detected. Fatigue and general malaise, nasal symptoms, and eye irritation typical in sick building syndrome were found in hotel staff. It can be seen that air quality and air quality management are related to diseases and their symptoms. ere are several factors involving indoor air quality. e environmental factors such as bioaerosol and human activity are also significant. Additionally, based on the risk assessment report of hospitals, the source of chemical emissions from wards is not considered. e study of relevant factors of indoor air quality management in hospitals is one approach to reduce hospital infections. erefore, this current study aimed at determining the airborne contamination levels in multiple-bed wards of hospitals, where more stringent hygiene standards are pursued. e purposes of this study were to determine concentrations of airborne microorganisms within a large hospital center in southern ailand. is study also compares the concentrations in different wards, relative to positions of electric fans, and by sampling time. Moreover, the physical environmental characteristics (temperature, humidity, and air velocity) and number of occupants expected to affect the concentrations of microorganisms were investigated. # Materials and methods ## Sample location. e hospital assessed in this study has a large ward capacity with 800 beds in total, and controlled natural ventilation includes windows, doors, and electric fans. Gola et al.informed that natural ventilation was identified as a strategy of healthier ventilation and natural force of wind-driven pressure through doors, apertures, and windows. e ventilation system of the ward was provided with wall fans for generating air motions. ere were 18 electric fans in each ward, with ambient air at 25.4-30.3°C and 72% relative humidity. e four wards were selected for male surgery (A), female surgery (B), male medicine (C), and female medicine (D) with 40 beds. e service hours were from 12:00 am to 8:00 pm. Sampling was done in three daily periods: before (10:00-12:00 am), during (4:00-6:00 pm), and after (8:00-10:00 pm) the patient visits. ## Sample collection. is study was performed from June to October, which was rainy season, in a hospital. Airborne microorganisms were collected in 3 points relating wind turbulence from electric fansto the positions of ceiling fans in the wards: under a ceiling fan, in the blow of a ceiling fan, and far away from the blow of ceiling fans (see. e sampling methods were bioaerosol sampling and open plate method. Each position was measured as follows: bioaerosol sampler at 60 cm height from floor level was set up to collect 100 liters and an open plate with culture medium was put at 60 cm height from floor level for 20 minutes. Trypticase Soy Agar (TSA) was used to cultivate bacteria and Malt Extract Agar (MEA) was used to cultivate fungi. e measurement of temperature and ventilation used a thermoanemometer (Model DF618) and WBGT Heat Stress Monitor was used to measure heat and humidity during each sampling period. e occupant density at the hospital was determined by observing the number of people within a period. e plates were incubated at 37°C for 24-48 hours and 48-72 hours for bacteria and fungi, respectively. Colony-forming units (CFUs) were counted and stained to identify the bacteria as Gram-positive or Gram-negative and to assess the morphology of microorganisms. # Data analysis. e airborne microorganism levels were analyzed from descriptive statistics and with t-tests. Multivariate analysis of variance (MANOVA) was carried out to explore group differences among the concentrations of bacteria and fungi by section wards, positions of electric fans, and time. When differences were detected, post hoc comparisons were performed using Tukey's honestly significant difference (HSD) to test the difference between different pairs of the groups. Differences were considered significant for p < 0.05. e associations among environmental characteristics with concentrations of bacteria and fungi were assessed from Pearson's correlation coefficients. # Results and discussion ## Concentrations of bacteria and fungi by hospital wards and various times. e average concentration of bacteria in bioaerosols sampler ranged from 335.5 to 928.89 CFU/m 3 whereas the open plate had the range 209.69-592.06 CFU/ m 3 . e highest average concentrations of bacteria were detected after the visiting hours in ward D, according to both measurement methods (see. Regarding the fungal concentrations, the average concentration from bioaerosols sampler method ranged from 134.50 to 487.22 CFU/m 3 , and for open plate measurements, the range was 18.82-87.50 CFU/m 3 . Also, the highest average concentrations of fungi were found in ward D when using bioaerosols sampler, but in ward A when using open plate at nighttime. Airborne microorganisms reached their concentration peak after the patient visits and then gradually decreased until the next visits. Our study revealed that the concentrations of airborne bacteria were higher than those of fungi, according to both sampling methods, agreeing in this sense with a prior study. Augustowska and Dutkiewiczindicated as monthly averages, measured in a hospital ward in Poland, 257-436 CFU/m 3 of bacteria and 10-96 CFU/m 3 of fungi. Our measurement results showed higher airborne concentrations than this prior study of a hospital ward in Poland. However, the measurement results of concentrations of bacteria did not exceed the standard limit of NIOSH set in 2004 and ACGIH (1,000 CFU/m 3 ), and the fungal concentration did not exceed the 500 CFU/m 3 of fungi guideline set by WHO in 2000. Higher airborne concentrations during nighttime were observed after the visiting hours (i.e., after 8:00 pm). Most microorganisms tend to come from skin (exudate) infected lesions and dust from human respiration, so the concentration of microorganisms tends to be higher indoors than outside of the building. Previous studies have reported greater concentrations indoors than outdoors, and our study agrees with this pattern. Ekhaise et al.and Jaffal et al.concluded that the indoor air environment can potentially place patients at greater risk than the outside environment because enclosed spaces can confine aerosols and allow their accumulation to reach infectious levels. e hospital wards have no housekeeping in the room areas after the service hours. is may increase infection risks for the patients. e level of cleanliness in the wards was considered. ## Comparisons of bacterial and fungal concentrations by hospital ward and by sampling time. e compared differences of bacterial and fungal concentrations at four different wards were statistically significant (p < 0.05), and the results are presented in. e results from biosampler indicate that the A, B, and C wards did not significantly differ in the mean bacterial concentrations, while ward D had significantly higher mean bacterial concentration than these. For fungi, wards A and D had significantly higher mean concentrations than wards B and C. e open plate sampling gave no significant differences in the mean concentrations of bacteria at B and D or between A and C. In the fungal concentrations, A and B did not differ, and neither did C and D. is is because of the same department on the same floor of the hospital, as well as proper ventilation systems. ere were significant differences in the mean fungal concentrations between the time periods (p < 0.05). e highest concentrations were measured during and after patient visits. Both bacterial and fungal concentrations were comparatively lower before the visiting time than at other observation periods. is might be because of the low night temperatures and high humidity. e high concentrations during and after visits could alternatively be attributed to the visitors. e results showed that there was no significant difference between the bacterial and fungal concentrations and the positions of electric fans. is study also revealed that the occurrence of air fluctuations and turbulence from electric fans did not affect exposure levels. e results disagree with the findings of a previous studyclaiming that microorganism contamination increased with the ambient air exchange rate when the fan blew air downwards. Air quality inside was very similar to air quality outside, indicating that there was good natural ventilation quality implemented for the building. ## Environmental characteristics and concentrations of airborne bacteria and fungi. e average temperature, air velocity, and humidity given by biosampler were. Temperature and humidity affect fungal concentrations significantly. Using the open plate data, a significant negative correlation was observed between the fungal concentrations and temperature. In addition, fungal concentrations were positively correlated with humidity given by biosampler. Normally, fungi grow the best in warmth and high humidity. In this study, patient rooms had a humid and warm climate, with a humidity of over 60% and a temperature of about 27°C. e high temperatures suggest that air conditioning was not much in use. It is probably too expensive, as it would be to dehumidify the air. e number of occupants, humidity, and temperature are physical factors associated with bacterial and fungal concentrations; however, these were not significantly affected by air velocity. Previous studies have indicated that the density of occupants is the key factor influencing the airborne concentrations in hospital wards. Our study agrees with these previous studies. Moreover, Zemouri et al.demonstrated that the daily cleaning operations in the hospital were related to the presence of aerosols. Normally, this area was cleaned twice per day (in the morning and in the afternoon). is cleaning frequency may not be a sufficient standard for safety. e management of cleaning procedures and the awareness of cleanings should be considered. Furthermore, a correlation between the number of people and the air volume was reported. Organizing patient visits in separate spaces, with distancing to prevent infections, and control of crowding seems reasonable. Further, for example, smartphones could be used for patient communications with family members during hospital visits. e results show that the fungal presence was very high, and aside from people, this may be attributed to air ventilation and air conditioning. Most of the bacteria related to patients can be released in a hospital environment and can be breathed in by visitors and staff. is study contrasted with the findings on temperature by Park et al.who reported that the temperature significantly influenced the concentration of bacteria. No relationships between bacterial or fungal concentrations and air velocity were found. ## Concentrations of gram-positive and gram-negative bacteria. Our results indicate that Gram-positive cocci and rod-shaped bacteria were present in significantly larger numbers than the Gram-negative bacteria throughout the experiment. It has been reported that Gram-positive bacteria are more tolerant to environmental stresses than Gramnegative bacteria. e cell walls of Gram-negative bacteria are thinner and less compact, whereas in Gram-positive cells peptidoglycan forms a thick compact cell wall, which is the outermost structure of Gram-positive cells. Airborne Gram-positive cocci are most frequently detected indoors in hospitals. e samples collected were analyzed by microscopy. e overall culturable fungi were Aspergillus spp., Penicillium spp., Cladosporium spp., Alternaria spp., and Curvularia spp. Aspergillus spp. was the most frequently identified species in this study, and these fungal pathogens are associated with airway diseases. It has been reported that Aspergillus spp. are the most common fungi found in various environments, such as food, water, and plants. Warmth with high moisture and lack of sunlight is favorable for their growth. A previous study reported that Aspergillus spp. caused aspergillosis and allergic aspergillosis syndrome. A study on concentrations of airborne microorganisms indicated that most fungi were found in a food center (Penicillium spp., Aspergillus spp., and Cladosporium spp.). However, these did not cause illnesses or health problems, possibly due to our immune system. Moreover, Penicillium spp. and Aspergillus spp. are common fungi found in buildings and are not considered pathogens. Nevertheless, these fungi might cause allergy, asthma, or respiratory illnesses. Hameed et al.also reported on the diversity of airborne bacteria and fungi in Helwan city of Egypt, where the fungi most found were Aspergillus, Cladosporium, and Penicillium, in this order. In addition, Aspergillus spp., Penicillium spp., Cladosporium spp., Curvularia spp., and Alternaria spp. were found indoors as Journal of Environmental and Public Health microbiological contaminants in the air, at a university building. Our results overall agree with these prior studies. Some particular fungi are linked with the sick hospital syndrome, causing allergenic diseases nonspecifically, and come from construction dust or dust accumulated within ventilation. Most invasive infections are acquired from indoor air. e prevention and control of microorganism growth can reduce the human health risks from airborne microorganisms. To reduce concentrations of airborne contaminants, it is necessary to implement some control measures in wards, such as using air purifiers and sterilization using ozone. # Conclusions Based on our measurements in a large hospital, located in southern ailand, the airborne bacterial concentrations were significantly higher than the fungal concentrations. e general air quality characteristics indicated that bacteria can grow and are more prevalent than mold. e airborne bacterial and fungal concentrations did not exceed the standard limits, respectively, set by the NIOSH and the WHO. is study also revealed that the occurrence of air fluctuations and turbulence from electric fans had no effects on the level of exposure. Air quality inside is very similar to air quality outside, indicating good natural ventilation quality in the building. e five types of fungi found were Aspergillus spp., Penicillium spp., Cladosporium spp., Alternaria spp., and Curvularia spp. In general, these fungi are mostly observed in high-risk areas such as schools, theatres, and hospitals. Exposure to these contaminants may affect the respiratory system. e results suggest that the number of occupants in a building is positively associated with the airborne bacterial concentrations. Additionally, high humidity and high temperature were observed in multiple-bed hospital wards. is might be because of the tropical climate in the area of this study. e quality of air in the wards was better early in the day than later after the morning has passed. Room cleaning should be performed after patient visits, to decrease airborne bacteria. Cleaning and housekeeping of all room areas need to be regular to decrease the airborne contamination in all areas for patients, visitors, and staff. Nosocomial infections with indoor air quality in the buildings will be investigated in related future studies. In further research on factors that affect indoor air quality, chemical factors should be considered. ## Data availability e data used to support the findings of this study are available from the corresponding author upon request.

Stochastic dynamics of macromolecular-assembly networks The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here, we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage k induction switches, which rely on the formation of DNA loops by proteins and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes.Subject Categories: metabolic and regulatory networks; computational methods # Introduction Cells consist of thousands of different molecular species that orchestrate their interactions to form extremely reliable functional units [bib_ref] From molecular to modular cell biology, Hartwell [/bib_ref]. Such molecular diversity and the pervasive ability of cellular components to establish multiple simultaneous interactions typically lead to the formation of large heterogeneous macromolecular assemblies, also known as complexes [bib_ref] Assembly of cell regulatory systems through protein interaction domains, Pawson [/bib_ref]. These complexes form the backbone of the most fundamental cellular processes, including gene regulation and signal transduction. Important examples are the assembly of the eukaryotic transcriptional machinery [bib_ref] Role of general and gene-specific cofactors in the regulation of eukaryotic transcription, Roeder [/bib_ref] , with about hundred components, and the formation of signaling complexes [bib_ref] Signaling complexes: biophysical constraints on intracellular communication, Bray [/bib_ref] , with tens of different molecular species. One of the main challenges facing modern biology is to move forward from the reductionist approach into the systemic properties of biological systems [bib_ref] Biological networks: the tinkerer as an engineer, Alon [/bib_ref]. A major goal is to understand how the dynamics of cellular processes emerges from the interactions among the different molecular components. Typical computational approaches approximate cellular processes by networks of chemical reactions between different molecular species [bib_ref] Modelling cellular behaviour, Endy [/bib_ref]. A strong barrier to this type of approaches is the inherent complexity of macromolecular complex formation. Complexes are typically made of smaller building blocks with a modular organization that can be combined in a number of different ways [bib_ref] Assembly of cell regulatory systems through protein interaction domains, Pawson [/bib_ref]. The result of each combination is a specific molecular species and should be considered explicitly in a chemical reaction description. Therefore, there are potentially as many reactions as the number of possible ways of arranging the different elements, which grows exponentially with the number of the constituent elements. Twenty components, for instance, already give rise to over a million of possible species. Two main types of avenues have been followed to tackle the exponential growth of the number of molecular species that arise during macromolecular assembly. One is based on computer programs that generate reaction rate equations for all of the macromolecular species [bib_ref] Computer-based analysis of the binding steps in protein complex formation, Bray [/bib_ref]. The other generates the different species dynamically [bib_ref] Automatic generation of cellular reaction networks with Moleculizer 1.0, Lok [/bib_ref]. Yet, none of the existing methods provides a consistent way to estimate the reaction rates. This obstacle is remarkable because the potential number of rates is even higher than the number of possible complexes. As a result, those methods often lead to unrealistic situations, such as the formation of polymeric complexes that do not exist under physiological conditions, which has been noted explicitly as intriguing caveats of the existing methodologies [bib_ref] Computer-based analysis of the binding steps in protein complex formation, Bray [/bib_ref] [bib_ref] Automatic generation of cellular reaction networks with Moleculizer 1.0, Lok [/bib_ref]. Here, we review the thermodynamic concepts underlying macromolecular complex assembly and examine how they can be used to both derive the dynamics of complex formation and estimate the transition rates needed to build a faithful computational model. The resulting stochastic approach does not give rise to the formation of unrealistic complexes and addresses the exponential growth of the number of species by stochastically exploring the set of representative complexes. In this way, it brings the properties of macromolecular interactions across scales up to the dynamics of cellular networks. To illustrate the applicability of this approach, we focus on DNA-protein complexes and their integration in gene regulatory networks. We consider as prototype systems the induction switches in the lac operon (Mü ller [bib_ref] Quality and position of the three lac operators of E. coli define..., Mü Ller-Hill [/bib_ref] and phage l [bib_ref] Four dimers of lambda repressor bound to two suitably spaced pairs of..., Ptashne [/bib_ref] , the two systems that led to the discovery of gene regulation [bib_ref] Genetic regulatory mechanisms in the synthesis of proteins, Jacob [/bib_ref]. ## Representation of macromolecular complexes A crucial aspect is to use a description for macromolecular complexes that can capture the underlying complexity in simple terms. It is possible to take advantage of the fact that macromolecular complexes have typically unambiguous structures, where only certain molecular species can occupy a given position within the complex. In such cases, the specific configuration or state of the macromolecular complex can be described by a set of M variables, denoted by s¼(s 1 ,ys i ,ys M ), whose values indicate whether a particular molecular component is present or not at a specific position. We chose s i ¼1 to indicate that the component is present and s i ¼0 to indicate that it is not present [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref]. With this description, the potential number of specific complexes is 2 M and the number of reactions 1 2 2 M (2 M À1)E2 2MÀ1 . The use of binary variables provides a concise method to describe all the potential complexes without explicitly enumerating them. This type of approach has been used in a wide range of interesting biological situations, such as diverse allosteric processes [bib_ref] Conformational spread: the propagation of allosteric states in large multiprotein complexes, Bray [/bib_ref] , binding of molecules to a substrate [bib_ref] Transition modes in Ising networks: an approximate theory for macromolecular recognition, Keating [/bib_ref] , binding of multistate proteins to receptor docking sites [bib_ref] Signaling through receptors and scaffolds: independent interactions reduce combinatorial complexity, Borisov [/bib_ref] , and signaling through clusters of receptors [bib_ref] Heightened sensitivity of a lattice of membrane receptors, Duke [/bib_ref]. In practice, current binary-variable approaches have strong limitations to tackle the assembly of macromolecular complexes. On the one hand, there are combinations of variables that do not have a physical existence. Explicitly, if a component that bridges two disconnected parts of the complex is missing, then the complex does not exist and if two components occupy the same position, they cannot be present within the complex simultaneously. On the other hand, the structure of the complex does not have to remain fixed. A complex can have different conformations and the components can be present in several states with different properties. None of the existing approaches based on binary variables incorporates all these key features needed to study macromolecular complex formation. In the following section, we exploit the underlying thermodynamics to put forward a binary description applicable to macromolecular assembly. ## Thermodynamics of assembly Thermodynamics allows for a straightforward connection of the binary description with the molecular properties of the system. Each configuration of the macromolecular complex has a corresponding free energy, which is a quantity that indicates the tendency of the system to change its state. Transformations that decrease the free energy of the system are favored over those that increase it; that is, the tendency of the system is to evolve towards the lowest free energy minimum. The statistical interpretation of thermodynamics [bib_ref] New York: C Scribner's Sons Gillespie DT (1976) General method for numerically..., Gibbs [/bib_ref] connects the equilibrium probability P s of state s with its free energy DG(s) through the expression [formula] P s ¼ 1 Z e ÀDGðsÞ=RT ; [/formula] where Z¼ P s e ÀDG(s)/RT is the normalization factor and RT is the gas constant times the absolute temperature (RTE 0.6 kcal/mol for typical experimental conditions). One crucial aspect is that the free energy of any given configuration, s¼(s 1 ,y,s i y,s M ), can be obtained to any degree of accuracy by expanding the free energy in powers of the binary variables: [formula] DGðsÞ ¼DGð0Þ þ X M i¼1 DG i s i þ X MÀ1 i¼1 X M j¼iþ1 DG ij s i s j þ X MÀ2 i¼1 X MÀ1 j¼iþ1 X M k¼jþ1 DG ijk s i s j s k þ Á Á Á [/formula] The first term on the right-hand side of the equation is the free energy of the empty complex, when none of their components is present, and it serves as a reference free energy; the second term takes into account the energetic cost of placing each element in the complex; the third term accounts for the pairwise interactions between elements; the fourth term accounts for interactions that need a third element to take place; and higher order terms, not shown in the equation, account for interactions that take place only when Representation and thermodynamics of macromolecular assembly. (A) An example of a macromolecular complex in one of eight possible states is used as illustration of the binary description. The complex consists of three molecular positions A, B, and C, described by binary variables s A , s B , and s C , respectively. In this case, A and C are occupied (gray shapes) and B is unoccupied (white shape with dashed contour). Red lines represent pairwise interactions between the components. This description can easily be connected to the thermodynamic properties of different configurations. Here, the free energy DG o for the configurations and their contributions are expressed in units of kcal/mol. The positional and interaction free energies are assumed to be 15 and À20, respectively. Note that the description refers to a three-molecule complex at a specific location. If, instead, one molecule is used as a reference, its positional free energy should not be counted. The free energy of the disconnected configuration (B) is much higher than the free energy of the connected configuration (C). These energetic considerations indicate that the disconnected configuration is extremely less abundant than the connected one. The stability of a compact structure (D) is considerably higher than that of a chain-like structure (C) because of the additional free energy of interaction. multiple elements are present. This expansion can be stopped at any degree of complexity. Typical network diagrams, such as those of protein-protein interactions, represent only up to pairwise interactions (the first three terms). The main advantage of this expansion is that the 2 M free energies needed to characterize all the possible configurations can be obtained from a much smaller subset. In order to connect the lower terms of this expansion directly with experimental data, the most important piece of information is that the free energy of binding, DG bind , between two elements can be decomposed into two main contributions : [formula] DG bind ¼ DG pos þ DG int : [/formula] One of them, the interaction free energy DG int , arises from the interactions between the two molecules, such as electrostatic, hydrophobic and Van der Waals interactions [bib_ref] Classical electrostatics in biology and chemistry, Honig [/bib_ref]. The other, the positional free energy DG pos , results from positioning the molecules in the right place and orientation so that they can interact and it accounts, among other potential contributions, for the loss of translational and rotational entropy upon binding [bib_ref] Entropic contributions to rate accelerations in enzymic and intramolecular reactions and chelate..., Page [/bib_ref]. The expression for the positional free energy can be written as Typical values of the molar positional free energy are DG pos o E15 kcal/mol [bib_ref] The price of lost freedom: entropy of bimolecular complex formation, Finkelstein [/bib_ref]. This value has been computed theoretically and measured experimentally for simple molecules [bib_ref] Entropic contributions to rate accelerations in enzymic and intramolecular reactions and chelate..., Page [/bib_ref] [bib_ref] The price of lost freedom: entropy of bimolecular complex formation, Finkelstein [/bib_ref]. For complex proteins, it might differ slightly. For instance, for the binding of AChE-Fas2, it has been estimated to be 9 kcal/mol [bib_ref] The entropic cost of protein-protein association: a case study on acetylcholinesterase binding..., Minh [/bib_ref]. Such high values of the positional free energy indicate that if the free energy of interaction is zero, the state in which two molecules are as close as if they were bound is extremely unlikely. Even small values of binding free energies, such as DG bind o EÀ2 kcal/mol, would imply considerably high interaction free energies, such as DG int EÀ17 kcal/mol (assuming DG pos o E15 kcal/mol). This is a crucial point in order to properly account for disconnected complexes. If the bridging element is missing, there is a missing positional free energy term and two missing interaction free energy terms [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref] and C). Thus, the free energy of the disconnected complex [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref] is much higher than that of the connected one [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref] , which indicates that the disconnected complex is extremely less abundant than the connected one under physiological conditions. The contribution of the positional free energy is also essential to avoid the presence of spurious polymeric complexes. A compact complex [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref] will have additional free energies of interaction compared to the chain-like one [fig_ref] Figure 1: Representation and thermodynamics of macromolecular assembly [/fig_ref] , which will render it much more stable than the polymeric chain. Finally, when two elements cannot be present in the same position at the same time, with this approach their interaction energy is infinitely large and the complex does not exist in practice. The term P i¼1 MÀ1 P j¼i þ 1 M DG ij s i s j accounts, among others, for interactions between components of the complex that have a multidomain structure, where domains interact in a pairwise manner with each other. In general, as we show in detail in the examples below, one should also consider the conformational free energy that accounts for the structural changes needed to accommodate multiple simultaneous interactions. This type of interactions requires higher order terms in the free energy expansion, such as P [formula] i¼1 MÀ2 P j¼i þ 1 MÀ1 P k¼j þ 1 M DG ijk s i s j s k . [/formula] All these thermodynamic concepts blend naturally into a binary-variable description to provide an approach that incorporates the key elements needed for studying macromolecular complex assembly, such as avoiding the formation of unrealistic polymeric complexes, taking into account disconnected complexes, considering conformational changes, and incorporating mediated or multicomponent interactions. The explicit way in which the thermodynamic approach is applied depends on the degree of characterization of the system. In the best-case scenario, all the required free energies are known and the behavior of the system can be predicted straightforwardly in full detail. If a few free energies are missing, it is possible to build a model based on the known interactions and obtain the unknown free energies by 'reversing the model' [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref]. The resulting free energies can then be used in new situations. This type of approach has been used, for instance, to infer for the first time the in vivo free energies of looping DNA by the lac repressor as a function of the length of the loop . It is also possible to use available sophisticated software packages to compute free energies from the known structures [bib_ref] Classical electrostatics in biology and chemistry, Honig [/bib_ref] [bib_ref] Electrostatics of nanosystems: application to microtubules and the ribosome, Baker [/bib_ref]. Such sophistication has not been achieved yet for computing reaction rates. On the other hand, when a substantial amount of information is missing, one can postulate interactions and use a thermodynamic approach to explore the potential types of behavior. In general, the thermodynamic approach needs quantitative inputs, but the requirements are much less stringent than those of other quantitative approaches based on chemical reactions. ## Applications: making networks out of complexes and vice versa The formation of DNA loops by proteins and protein complexes in the regulation of the lac operon and phage l provides challenging examples to illustrate the applicability of this approach. Full understating of these two genetic systems requires the use of thermodynamic concepts not considered by current methods to study macromolecular assembly. These concepts are essential to tackle more complex situations, such as gene regulation in eukaryotes, which relies widely on DNA looping to implement action at a distance from regulatory elements that are far away from the promoter region [bib_ref] The eukaryotic transcriptional machinery: complexities and mechanisms unforeseen, Roeder [/bib_ref] [bib_ref] DNA-looping by RXR tetramers permits transcriptional regulation 'at a distance', Yasmin [/bib_ref]. In particular, decomposition of the free energy into interaction, positional, and conforma-tional contributions is crucial for understanding how weak distal DNA binding sites can strongly affect transcription [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref] and how proteins that do not oligomerize in solution do so on DNA to form DNA loops [bib_ref] Octamerization of lambda CI repressor is needed for effective repression of P(RM)..., Dodd [/bib_ref] [bib_ref] DNA looping in gene regulation: from the assembly of macromolecular complexes to..., Vilar [/bib_ref]. Hereafter, to simplify the notation, we will refer to positional, interaction, and conformational free energies by p, e, and c, respectively. One remarkable aspect of our approach is that the binaryvariable method we propose has an equivalent representation in terms of network diagrams, in which nodes indicate whether or not a component or molecular conformation is present and links represent the interactions among components and molecular conformations. These networks can be directly mapped to the underlying macromolecular structure of the complexes and their properties, as illustrated in detail below for the cases of DNA looping in the lac operon and phage l. The resulting networks strikingly resemble the underlying molecular structures because nodes are associated with properties of the components, which have a determined arrangement in space. In this type of interaction networks, the whole network specifies a single state of the system, in the same way as a state of the macromolecular assembly is specified by indicating where each component is within the complex. This component-oriented description allows for a representation that is not affected by the exponential explosion in the number of states as the number of components increases. In other widely used quantitative methods with network representations, like Markov Chains, each node represents a state of the system and therefore there are as many nodes in the network as potential states. In those state-oriented networks, only one node is occupied at a time and the behavior of the system is represented by a series of jumps from one node to another. In our case, several nodes can be occupied simultaneously and the behavior of the system is given by the sequence of changes in occupancy. In [fig_ref] Figure 2: Interaction networks [/fig_ref] , we compare a general interaction network representation for a three-component complex with the graphical representation of a Markov Chain for the same system. The number of nodes in the interaction network is the same as the number of components. In the Markov Chain network, in contrast, the number of nodes is 2 to the number of components. There are also qualitative graphical representations, like 'Molecular Interaction Maps' [bib_ref] Molecular interaction maps of bioregulatory networks: a general rubric for systems biology, Kohn [/bib_ref] and 'process diagrams' [bib_ref] Using process diagrams for the graphical representation of biological networks, Kitano [/bib_ref] , which aim at describing many types of cellular and biochemical processes that extend beyond macromolecular assembly. They can be translated into computational models, but how to perform that translation is not given explicitly by the representation and typically depends on the details of the processes involved. As a result, although the representation itself might not be affected by an exponential increase in the number of states [bib_ref] Molecular interaction maps of bioregulatory networks: a general rubric for systems biology, Kohn [/bib_ref] , the resulting mathematical description might be susceptible to this problem. A key difference with our approach is therefore that our representation gives straightforwardly the precise quantitative behavior of the system from its equivalence with the equations that describe the macromolecular assembly. This avenue for incorporating binary variables with thermodynamics also presents important differences with the way in which thermodynamic concepts have been used so far in macromolecular assembly. Thermodynamic concepts applied to gene regulation were pioneered by Shea and Ackers [bib_ref] Quantitative model for gene regulation by lambda phage repressor, Ackers [/bib_ref] [bib_ref] The OR control system of bacteriophage lambda. A physical-chemical model for gene..., Shea [/bib_ref] and subsequently used in a variety of situations [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref] [bib_ref] Transcriptional regulation by the numbers: applications, Bintu [/bib_ref] [bib_ref] DNA looping in gene regulation: from the assembly of macromolecular complexes to..., Vilar [/bib_ref]. In the traditional framework, the probability for the system to be in a state k is given by [formula] P k ¼ 1 Z ½N j k e ÀDG o k =RT ; [/formula] where DG k o is the standard (molar) free energy of the state k, j k is the number of molecules bound in the state k, and Z ¼ P k ½N j k e ÀDG o k =RT is the normalization factor. The summation is taken over all the states. Thus, to describe the system with the traditional approach one has to give the standard free energy for each state as well as the number of molecules bound. Typically, this is done in the form of a table, which has as many entries as the number of states of the system. Thus, for a system with three components, there is a table with eight entries for the standard free energy and another eight for the number of molecules. In the approach we have proposed, there is a simple formula that accounts for the free energy of all the states, and the resulting expressions have a compact form amenable to computational and mathematical manipulations. As we show in the following sections, this approach brings forward explicitly the connection between macromolecular structure and function and its integration into the biochemical dynamics of cellular networks. Interaction networks. The state and properties of the macromolecular structure can be described by an interaction network. Nodes (big gray circles) in the interaction network represent whether or not a component is present. Small black circles are joined to nodes and represent interactions between the elements they join. Labels associated with black circles indicate the contributions to the free energy arising when all the nodes they are linked to are occupied. This graphical representation is equivalent to the mathematical expression of the free energy in terms of binary variables. For the network shown here, the free energy of a state s¼(s A ,s B ,s C ) is given by DG The lac operon [formula] (s)¼ p(s A þ s B þ s C ) þ e AB s A s B þ e BC s B s C þ e CA s C s A , [/formula] The lac operon consists of a regulatory domain and three genes required for the uptake and catabolism of lactose (Mü ller [bib_ref] Quality and position of the three lac operators of E. coli define..., Mü Ller-Hill [/bib_ref]. Its main regulator is the lac repressor, which can bind to DNA at the main operator site O1, thus preventing transcription of the genes, and to an auxiliary operator (O2 or O3) by looping the intervening DNA. For a system with two operators (O1 and O2), the lac repressor-DNA complex [bib_ref] Crystal structure of the lactose operon repressor and its complexes with DNA..., Lewis [/bib_ref] can be in five representative states [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref] : (i) none of the operators is occupied, (ii) a repressor is bound to just the auxiliary operator, (iii) a repressor is bound to just the main operator, (iv) a repressor is bound to both the main and the auxiliary operators by looping the intervening DNA, and (v) one repressor is bound to the main operator and another repressor, to the auxiliary operator. We can express the free energy of all these states in a compact form in terms of binary variables: [formula] DGðsÞ ¼DGð0Þ þ pðs 1 þ s 2 Þ þ p DNA s DNA þ ðe 1 s 1 þ e 2 s 2 Þs DNA þ ðc L À ps 1 s 2 Þs L s DNA ; [/formula] where s 1 and s 2 are the binary variables that indicate whether (s i ¼1, for i¼1, 2) or not (s i ¼0, for i¼1, 2) the repressor is bound to O1 and O2, respectively; s DNA indicates the presence (s DNA ¼1) or absence (s DNA ¼0) of DNA; and s L is a variable that indicates the molecular conformation of DNA, either looped (s L ¼1) or unlooped (s L ¼0). The quantities p and p DNA are the positional free energies of the repressor and DNA, respectively; e 1 and e 2 , the interaction free energy between the repressor and O1 and O2, respectively; and c L , the conformational free energy of looping DNA. DG(0) is the free energy of the reference state in which there is no repressor, DNA, or DNA looping (all the binary variables are zero). We are interested in the binding of the repressor to DNA and therefore DNA is always present (s DNA ¼1), which simplifies the description to just three binary variables: DGðsÞ ¼ ðp þ e 1 Þs 1 þ ðp þ e 2 Þs 2 þ ðc L À ps 1 s 2 Þs L ; where we have chosen DG(0)¼Àp DNA so that the reference free energy is equal to zero when no repressor is bound and there is no DNA looping. The network representation of the lac repressor-DNA assembly with the three binary variables [fig_ref] Figure 3: Repressor-DNA assembly and gene regulation in the lac operon [/fig_ref] has a close connection with the underlying molecular structural properties [fig_ref] Figure 3: Repressor-DNA assembly and gene regulation in the lac operon [/fig_ref] , in such a way that, given the structural arrangement of a complex, our approach provides a straightforward avenue to obtain a network representation with an associated thermodynamic description. Explicitly, each node in the interaction network corresponds to a binary variable in the equation for the free energy. The decomposition of the free energy in its different contributions (positional, interaction, and conformational) becomes crucial to perform an expansion in terms of binary variables. From the statistical thermodynamics point of view, the binding of the repressor to two operators has only five relevant states (i-v). With the binary description of the state of the complex, there are three variables and therefore 2 3 ¼8 states. These two descriptions are in fact equivalent because three of the eight states in the binary description have significantly high free energies, that is, extremely low probabilities, which for practical purposes makes them irrelevant (see [fig_ref] Figure 3: Repressor-DNA assembly and gene regulation in the lac operon [/fig_ref]. The high free energies arise because for these states positional and conformational free energies are not balanced by interaction free energies. This approach can naturally be used to study the consequences that DNA looping has in gene regulation. In the lac operon, transcription takes place only when the main operator O1 is free; that is, when the binary variable s 1 is zero. Thus, the transcription rate, t, is proportional to the average value of 1 minus s 1 : [formula] t ¼ 1 Z X s t max ð1 À s 1 Þe ÀDGðsÞ=RT ; [/formula] where t max is the maximum transcription rate. This model shows a precise agreement with experiments [bib_ref] Quality and position of the three lac operators of E. coli define..., Mü Ller-Hill [/bib_ref] over the three orders of magnitude of the measured repression levels [fig_ref] Figure 3: Repressor-DNA assembly and gene regulation in the lac operon [/fig_ref]. The specific form that DNA looping confers to the repression level as a function of the repressor concentration has two notable characteristics. The repression level has both a significantly high value and a relatively flat profile around physiological lac repressor concentrations (B15 nM). Both properties have important general consequences for the underlying microbiochemistry of the cell. If concentrations of the different molecular species are kept low to prevent nonspecific interactions, not only is the binding to the specific sites decreased but also fluctuations are expected to become important [bib_ref] Stochastic gene expression in a single cell, Elowitz [/bib_ref] [bib_ref] Summing up the noise in gene networks, Paulsson [/bib_ref] [bib_ref] Gene regulation at the single-cell level, Rosenfeld [/bib_ref]. In the case of the lac operon, the average number of repressors per cell is very low, around 10, and, because of this low value, is expected to fluctuate strongly from cell to cell. The effects of DNA looping, as exemplified by the repression level, not only increase specificity and affinity of the lac repressor for the main operator but, at the same time, also make transcription fairly insensitive to fluctuations in the number of repressors. We can address a more general situation, which includes the binding of different molecular species at identical positions within the complex. To illustrate this possibility, we consider the case of mutant lac repressors that do not tetramerize [bib_ref] The three operators of the lac operon cooperate in repression, Oehler [/bib_ref]. In its dimeric form, the lac repressor has just a single DNA-binding domain and thus cannot loop DNA. In this case, the free energy is given by DG(s)¼(p d þ e 1 )s 1d þ (p d þ e 2 )s 2d , where p d is the positional free energy of the dimeric lac repressor and s 1d and s 2d are the two binary variables that account for its binding to O1 and O2, respectively. If this mutant repressor is expressed together with the wild-type (WT) lac repressor, it will compete for the binding to the operator sites and the free energy will be given by the sum of the free energies for WT and mutant repressors plus a contribution accounting for the interaction between the two types of repressors: DGðsÞ ¼ðp þ e 1 Þs 1 þ ðp þ e 2 Þs 2 þ ðc L À ps 1 s 2 Þs L þ ðp d þ e 1 Þs 1d þ ðp d þ e 2 Þs 2d þ 1ðs 1 s 1d þ s 2 s 2d Þ: The term N(s 1 s 1d þ s 2 s 2d ) introduces an infinite free energy of interaction when the two types of repressors are bound to the same operator simultaneously, thus making the probability of such states zero. The five-binary-variable description of the WT and mutant lac repressor-DNA complex formation is a straightforward extension of the one for just WT lac repressor. This example clearly illustrates how it is possible to use our approach to add complexity without escalating into an exponentially growing description. ## Phage k The genetic regulation of phage l provides an explicit example in which the DNA loop is formed not by a single protein, as in the lac operon, but by a protein complex that is assembled on DNA as the loop forms. The lysogenic to lytic switch in phage linfected Escherichia coli lysogens is controlled at two operators in the phage DNA. These two operators, known as the left, O L , and right, O R , operators, are located 2.4 kb away from one another. Each of them has a tandem of three DNA sites where phage l cI repressors can bind as dimers (cI 2 ): r1, r2, and r3 for the right operator, and l1, l2, and l3 for the left operator. Two cI dimers bound to r1 and r2 on the right operator can form an octamer with two cI dimers bound to l1 and l2 on the left operator by looping the intervening DNA. Stability of the E. coli lysogens is accomplished by repression of transcription by the phage l cI repressor of the cro gene at the P r promoter and regulation of its own transcription at the P rm promoter. Explicitly, binding of cI repressor dimers to r2, when r3 is vacant, activates its own transcription. When r3 is occupied, cI transcription is turned off. For a long time, one of the main puzzles in the regulation of phage l was that the strength of r3 was too weak for it to be occupied by cI 2 at the observed physiological concentrations [bib_ref] Four dimers of lambda repressor bound to two suitably spaced pairs of..., Ptashne [/bib_ref]. The missing element was that the formation of the DNA loop by the octamerization of the cI repressor dimers bound at r1, r2, l1, and l2 can bring l3 close to r3 so that the cI repressor can bind cooperatively as a tetramer to these two sites even though they are B2.4 kb apart. Modeling of this system already gets close to the limits of the traditional thermodynamic approach. The number of states is 128, which accounts for all the combinations This subtraction of a positional free energy in the looped state accounts for the fact that the simultaneously binding of a single repressor to both operator sites should include in the free energy two interaction terms and just one positional term. The free energies used here have been obtained from [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref]. To convert from the in vivo natural units (molecules/cell) to the more common biochemical ones (concentration), we have used 1 molecule/cell¼1.5 nM. The looping free energy has been computed as described in the caption of in [bib_ref] DNA looping in gene regulation: from the assembly of macromolecular complexes to..., Vilar [/bib_ref] with DG pos o ¼15 kcal/mol. (C) The free energies DG(s) (in kcal/mol) of the eight different states of the macromolecular network representation of the lac operon as a function of the lac repressor concentration indicate that the free energy of three states [s¼(0, 0, 1), (0, 1, 1), and (1, 0, 1), with s¼(s 1 , s 2 , s L )] is too high and that only the other five states play a relevant role. (D) The repression level (R) as a function of the lac repressor concentration for one (green circles and dashed lines) and two (red squares and continuous lines) operators shows an excellent agreement with the available experimental data. The values computed with R¼[t/t max ] À1 ¼[ 1 Z P s (1Às 1 )e ÀDG(s)/RT ] À1 (lines) are compared with the experimental data (symbols) from [bib_ref] Quality and position of the three lac operators of E. coli define..., Mü Ller-Hill [/bib_ref] at two repressor concentrations for three different strengths of the main operator O1. of occupancies of the six binding sites in either the looped or unlooped conformations of DNA. In terms of binary variables, however, the free energy of all the possible states of the assembly of the cI repressor-DNA complex is concisely described by [formula] DGðsÞ ¼ X 3 i¼1 ðp þ e ri Þs ri þ e r23 s r2 s r3 þ e r12 s r1 s r2 þ e r123 s r1 s r2 s r3 þ X 3 i¼1 [/formula] ðp þ e li Þs li þ e l23 s l2 s l3 þ e l12 s l1 s l2 þ e l123 s l1 s l2 s l3 þ ðc L þ e T s r3 s l3 þ e O s r2 s l2 s r1 s l1 Þs L : Here, p is the positional free energy of the cI repressor dimers; and e ri and e li , with i¼1, 2, 3, are the interaction free energy of the cI dimer with each of its three DNA binding sites at the right and left operators, respectively. The terms of the type e r23 s r2 s r3 account for the pairwise interactions of cI dimers bound at neighboring DNA sites. The terms with three binary variables, such as e r123 s r1 s r2 s r3 , are introduced because these pairwise interactions are affected by the binding of cI repressors to the other neighboring site. The term (c L þ e T s r3 s l3 þ e O s r2 s l2 s r1 s l1 )s L accounts for the effects of DNA looping. It includes looping (c L ), cI tetramerization (e T ), and cI octamerization (e O ) contributions to the free energy. This system has a network representation with seven binary variables that closely follows from its molecular organization . As shown here, the seven-binary-variable description is equivalent to previous modeling of this system using the traditional thermodynamic approach [bib_ref] Cooperativity in long-range gene regulation by the lambda CI repressor, Dodd [/bib_ref]. It is important to note that the expression, e r23 s r2 s r3 þ e r12 s r1 s r2 þ e r123 s r1 s r2 s r3 , can be rewritten in the more familiar cI repressor-DNA complex formation and its effects on the activity of the P rm promoter in phage l. (A) The network representation of phage l cI dimers binding to DNA is displayed together with (B) a cartoon of the looped configuration with six cI dimers (in green) bound to looped DNA (in orange) and the P rm and P r promoters. cI repressor dimers can bind to any of the three sites on the right (r1, r2, and r3) and on the left (l1, l2, and l3) operator, with positional free energy p¼15À0.6 ln[N] kcal/mol and interaction free energies e ri and e li , with i¼1, 2, 3 for the right and left operators, respectively. The values used, in kcal/mol, are e r1 ¼À27.7, e r2 ¼À25.7, e r3 ¼À25.2, e l1 ¼À28.8, e l2 ¼À27.1, and e l3 ¼À27.4. In addition, if two cI dimers are bound to two consecutive sites at the right or left operators, there is a cooperative free energy of interaction between dimers, in units of kcal/mol, of e r12 ¼À3.0 (r1 and r2) and e r23 ¼À3.0 (r2 and r3) (right operator) and of e l12 ¼À2.5 (l1 and l2) and e l23 ¼À2.5 (l2 and l3) (left operator), represented by the lines connecting the neighboring pairs of sites. These two terms have to be compensated in the case that there is one dimer bound to each of the three sites in the left and/or right operators. This is indicated by the line connecting the three sites at each operator with an interaction free energy term of e r123 ¼3.0 kcal/mol (right) and e l123 ¼2.5 kcal/mol (left), and as the diagram indicates, this only happens when the three binary variables are s r1 ¼s r2 ¼s r3 ¼1 or s l1 ¼s l2 ¼s l3 ¼1. The additional binary variable s L indicates whether DNA is looped, which contributes to the free energy with c L ¼21 kcal/mol (free energy of DNA looping). DNA looping can mediate interactions between the dimers bound at the left and right operators and, thus, if s r1 ¼s r2 ¼s l1 ¼s l2 ¼1, the cI dimers at both operators can interact and form an octamer, with a contribution to the free energy of the complex of e O ¼À21.5 kcal/mol, and if s r3 ¼s l3 ¼1, the two dimers can interact and form a tetramer with an additional free energy contribution of e T ¼À3.0 kcal/mol. The values of the interaction free energies have been taken from [bib_ref] Cooperativity in long-range gene regulation by the lambda CI repressor, Dodd [/bib_ref] and modified slightly to improve the agreement with the experimental data. The octamer interaction and looping free energies have been chosen so that c L þ e O ¼À0.5 kcal/mol and their precise value does not affect the results provided that the preceding relationship between them holds. DG(s) is the expression of the free energy of the complex in terms of the seven binary variables. (C) The activity of the P rm promoter as a function of the cI dimer concentration is obtained from A¼ 1 Z P s (t act s r2 þ t bas )(1Às r3 )e ÀDG(s)/RT , with t bas ¼45 and t act ¼t actnl (1Às L ) þ t actl s L , with t actnl ¼420 for unlooped DNA and t actl ¼200 for looped DNA [bib_ref] Cooperativity in long-range gene regulation by the lambda CI repressor, Dodd [/bib_ref]. The cI 2 concentration of WT lysogens, [N lys ]¼2  10 À7 M, is used as reference. The computed activities (full lines) are compared with the experimental data (red symbols) from [bib_ref] Cooperativity in long-range gene regulation by the lambda CI repressor, Dodd [/bib_ref] for the WT (squares) system and two other cases: one with a weak l3 binding site with e 0 l3 ¼À21 kcal/mol (triangles) and the other with a large free energy of looping (c L ¼N), equivalent to a system with no left operator (circles). [formula] +(c L +e T s r3 s l3 +e O s r2 s l2 s r1 s l1 )s L 3 i=1 3 i=1 L P rm P r [N] / [N lys ] [/formula] form: e r23 s r2 s r3 (1Às r1 ) þ e r12 s r1 s r2 (1Às r3 ) þ (e r123 þ e r12 þ e r23 ) s r1 s r2 s r3 , which indicates that the free energy of the cooperative interactions between all the cI dimers bound to the right operator is e r123 þ e r12 þ e r23 . This expression explicitly reveals e r123 as the correction to the free energy arising from the effects of a third cI dimer on the intraoperator interactions between pairs of cI dimers and illustrates how it is possible to expand the free energy of a given configuration, s¼(s 1 ,ys i ,ys M ), in powers of the binary variables. In general, there is the potential for establishing multiple loops. In the traditional approach, considering two additional loops will increase the number of states from 128 to 256. In our case, this extension involves adding just three terms, [formula] (c L2 þ e O s r2 s l3 s r1 s l2 )s L2 þ (c L3 þ e O s r3 s l2 s r2 s l1 )s L3 þ N(s L s L2 þ s L s L3 þ s L2 s L3 ) [/formula] , to the free energy DG(s). Here, s L2 and s L3 are the binary variables for the two additional loops and c L2 and c L3 are the corresponding free energies of looping. For the WT situation, the probability of having these extra loops is very small and they do not significantly affect the behavior of the system [bib_ref] Cooperativity in long-range gene regulation by the lambda CI repressor, Dodd [/bib_ref]. However, they might become important in mutants with altered binding. The effects of the left operator and DNA looping in the induction switch of phage l are apparent in the transcription rate at the P rm promoter, which depends strongly on the occupancy of r2 and r3. There is transcription at a basal level t bas when neither r2 nor r3 are occupied and at an activated level t act when r2 is occupied and r3 is free. The activated transcription rate, in turn, depends on whether (t act ¼t actl ) or not (t act ¼t actnl ) DNA is looped [bib_ref] Interaction at a distance between lambda repressors disrupts gene activation, Hochschild [/bib_ref]. These dependences are expressed mathematically through [formula] t ¼ 1 Z X s ððt actnl ð1 À s L Þ þ t actl s L Þs r2 [/formula] þ t bas Þð1 À s r3 Þe ÀDGðsÞ=RT : The activity of P rm as a function of the cI repressor concentration shows a sharp maximum for WT phage DNA and a plateau-like maximum for two mutants in which the r3 site is not occupied at WT concentrations . One of these mutants has only the right operator and the other has a weak l3 site. The narrower maximum of WT allows for tighter control of the cI 2 concentration, whose production sharply decreases for high concentrations. This marked decrease is the result of the extra layer of cooperativity of binding to r3 introduced by DNA looping and has important consequences for the kinetics of the system. ## Stochastic dynamics and macromolecular assembly networks A relationship between the kinetics and the thermodynamic properties of the system can be exploited to infer transition rates. It is known as the principle of detailed balance and results from the fact that at equilibrium the rates of going from state s to state s 0 and its inverse, from s 0 to s, are the same. Mathematically, it implies P s k s-s 0 ¼P s 0 k s 0 -s , where P is the probability of the state denoted by its subscript and k is the transition rate of the processes denoted by its subscript. This expression, together with the equilibrium values of the probabilities, P s /P s 0 ¼e À(DG(s)ÀDG(s 0 ))/RT , leads to the following relationship between the probability transition rate constants between two states: k s!s 0 ¼ k s 0 !s e ÀðDGðs 0 ÞÀDGðsÞÞ=RT : The remarkable property of this expression is that reactions with known rates can be used to infer the rates of more complex reactions from the equilibrium properties. For instance, the association rate of many regulatory molecules to different DNA sites is practically independent of the particular DNA sequence. The dissociation rate, in contrast, strongly depends on the sequence. In this case, knowing one association rate can be used to obtain the dissociation rates for different binding sites through the principle of detailed balance. The inferred rates can then be used to study the dynamics of the system. The dynamics can be simplified further by following a procedure similar to that considered previously for the free energy: it is also possible to perform an expansion for the kinetics of the system, but now in terms of the number of components that can change simultaneously in a transition. We discuss in detail the case in which only one component can change at a given time: either the component gets into or out of the complex. For each component i, we can define on (k on i ) and off (k off i ) rates for the 'association' and 'dissociation' rates, respectively, which in principle will depend on the pre-and post-transition states of the complex. The explicit dynamics can be obtained by considering the change in binary variables as reactions s i ! ð1 À s i Þ with rates r i ¼ ð1 À s i Þk i on ðsÞ þ s i k i off ðsÞ: The reaction changes the variable s i to 1 when it is 0 and to 0 when it is 1, representing that the element gets into or out of the complex. The mathematical expression of the transition rate reduces to k on i when the element is outside the complex (s i ¼0) and to k off i when the element is inside the complex (s i ¼1). Typically, the on rate does not depend as strongly on the state of the complex as the off rate. The on rate is essentially the rate of transferring the component from solution to the complex. The off rate, in contrast, depends exponentially on the free energy. The principle of detailed balance can be used to obtain the off rates from the on rates: k i off ðsÞ ¼ k i on e ÀðDGðs 0 ÞÀDGðsÞÞ=RT : These remarkably compact expressions for the transition rates between different states of the complex can be considered together with other reactions that affect or depend on the state of the complex. In this way, it is possible to integrate the stochastic dynamics of macromolecular assembly into networks of chemical reactions and move the effects of macromolecular assembly up to the properties of cellular processes. The stochastic dynamics of the resulting networks of reactions and transitions can then be obtained with well-established Monte-Carlo algorithms [bib_ref] New algorithm for Monte-Carlo simulation of ising spin systems, Bortz [/bib_ref]. ## Phage k ci repressor self-regulation We illustrate the integration of macromolecular assembly into the dynamics of cellular processes through the kinetics of phage l cI repressor's self-regulation feedback. The traditional approach to simulate the dynamics of this system would have to consider all the 128 states of the cI-DNA complex as chemical species. Therefore, considering just the kinetics of binding, unbinding, and looping would involve 128 rate equations, one for each state. In addition to writing down these equations, the traditional approach would need as inputs the values of the 8128 rates that connect the 128 states with each other. The onerous complexity of the resulting procedure has prevented so far the simulation of the stochastic induction switch with DNA looping. There are kinetic studies only for the induction without DNA looping [bib_ref] Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli..., Arkin [/bib_ref]. With the approach we have developed, the simulation of the stochastic kinetics of phage l cI repressor's self-regulation feedback follows straightforwardly. Our graphical representation, which accounts in full quantitative detail for the assembly of macromolecular complexes, can be seamlessly integrated with the canonical textbook representation of chemical reactions [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref]. In principle, this type of integration is also possible with qualitative representations like 'Molecular Interaction Maps' [bib_ref] Molecular interaction maps of bioregulatory networks: a general rubric for systems biology, Kohn [/bib_ref] and 'process diagrams' [bib_ref] Using process diagrams for the graphical representation of biological networks, Kitano [/bib_ref] to provide them with a precise stochastic dynamics. We have considered the cI-DNA complex together with the different stages of protein production from transcription at the promoter to protein dimerization for active repressors to study the stochastic dynamics of the phage l repressor self-regulation. The on rates of the different transitions are k on i ¼a[N] for i¼r1, r2, r3, l1, l2, and l3 (describing binding to the DNA operators by cI repressor dimers, cI 2 ) and k on i ¼b for i¼L (DNA looping), with a and b constants. The off rates follow from the detailed balance principle, as delineated previously. In addition, we consider the following chemical reactions (see [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref] legend for details): cI mRNA production and degradation; cI repressor production and degradation; cI repressor dimerization and cI 2 dissociation; and cI 2 nonspecific binding and degradation. The rate of cI mRNA production, k t , is a function of the state of the macromolecular complex, as described previously: k t ¼(k act s r2 þ k bas )(1Às r3 ), where k act and k bas are the activated and basal cI mRNA production rates. This self-regulatory network incorporates the macromolecular complexity at the promoter region as a module [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref]. Only a few of the elements of the DNA-protein complex are directly coupled to the cellular dynamics. Specifically, there is an input, the cI dimer concentration ([N]), and two main outputs, the occupancy of the r3 and r2 sites, which control the production of cI mRNA (k t ). Depending on the free energy of DNA looping, this module has different behaviors (see [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref]. For a high free energy of DNA looping (green curves), so that it is very difficult to form the loop, the steady state cI 2 concentration [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref] is relatively high and noisy in the lysogenic state, when the degradation rate of cI is low [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref]. In contrast, for free energies of DNA looping close to WT levels (red curves), cI 2 concentration in the lysogenic state is tightly regulated and remains narrowly constrained at low values, exhibiting small fluctuations. Quantitatively, the fluctuations of concentration, usually referred to as noise, are characterized by the variance divided by the mean of the number of molecules: Z¼ (/N 2 SÀ/NS 2 )//NS [bib_ref] Stochastic gene expression in a single cell, Elowitz [/bib_ref]. In the lysogenic state, DNA looping substantially lowers the strength of the intrinsic noise, from values of Z¼7.9, when there is no DNA looping, to Z¼2.7. Switching from the lysogenic to the The phage l cI-DNA assembly network can we viewed as a module within the network that controls the production of cI repressors at the P rm promoter. Binding of cI dimers (cI 2 ) to r3 (s r3 ¼1), represses transcription, whereas binding to r2 when r3 is unoccupied (s r2 ¼1 and s r3 ¼0) activates transcription. The transcription rate is given by k t ¼(k actnl (1Às L )s r2 þ k actl s L s r2 þ k bas )(1Às r3 ), with k actnl ¼0.05 s À1 , k actl ¼ 0.0225 s À1 , and k bas ¼0.005 s À1 ; cI mRNA is degraded and translated into cI protein at rates k mdeg ¼0.005 s À1 and k p ¼0.05 s À1 per mRNA, respectively; and cI monomers and dimers are degraded at a rate k deg . The cI dimerization reaction takes place with association and dissociation rate constants k a ¼7  10 8 M À1 s À1 and k dis ¼9.9 s À1 , respectively. cI dimers in solution can bind nonspecifically to DNA, with an equilibrium binding constant K ns ¼2.0  10 4 M À1 , or bind to one of the free operators with an association rate k on ¼a[N], with a¼7  10 8 M À1 s À1 . Note that the values used for the association rates are the typical ones of diffusionlimited reactions. The dissociation rates k off depend on the free energy difference between the complexes with (s) and without (s 0 ) the cI dimer via the detailed balance principle: k off ¼k on e À(DG(s 0 )ÀDG(s))/RT . As cellular volume, we have taken 10 À15 l. The rates of looping and unlooping DNA are k on L ¼30 s À1 [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref] and k off L , respectively, with k off L obtained also from the detailed balance principle. (B) Time behavior of the number of cI dimers for the case with WT operators (red) and with no left operator (green) when (C) the cI degradation rate k deg (in units of min À1 ) is switched from 0.025 min À1 to 0.4 min À1 . Time on the horizontal axis is given in hours. (D) Activity of the promoter P r controlling a reporter gene. In this case, there is transcription at a rate k t rep ¼ 0.12(1Às r1 )(1Às r2 ) s À1 only when both r1 and r2 are free. The reporter mRNA translation, mRNA degradation, and protein degradation rates are k p rep ¼ 0.01 s À1 , k mdeg rep ¼0.005 s À1 , and k deg rep ¼0.005 s À1 , respectively. lytic states happens as the degradation rate of cI is sharply increased. After the switch, cI 2 concentration goes to almost zero in both cases. The concentration of cI 2 also affects another important output of the cI 2 -DNA assembly module: the occupancy of r1 and r2. This output controls the P r promoter, which leads to transcription at a given rate when r1 and r2 are free and to no transcription when r1 or r2 are occupied. The activity of a reporter gene controlled by this promoter does not show a marked dependence on the free energy of looping [fig_ref] Figure 5: Phage l cI self-regulation kinetics [/fig_ref]. Therefore, DNA looping allows for tight control of cI 2 concentration at low levels without substantially affecting the turning on and off of genes at the P rm and P r promoters. Remarkably, it has recently been found experimentally that turning on transcription of the P r promoter by increasing the degradation of cI 2 is not affected by the presence of DNA looping [bib_ref] On the role of Cro in lambda prophage induction, Svenningsen [/bib_ref]. As in the case of the lac operon, here DNA looping makes the system function reliably with low numbers of molecules. ## Conclusions: from molecules to networks The study of cellular processes requires a balance of scope and detail. Whereas single molecular interactions can be modeled in full atomic detail with current technologies [bib_ref] Computer simulation studies of model biological membranes, Saiz [/bib_ref] [bib_ref] Molecular dynamics and protein function, Karplus [/bib_ref] , approximations in terms of chemical reactions are needed when turning to processes that reach the cellular scale and involve networks of interacting molecular species [bib_ref] Modelling cellular behaviour, Endy [/bib_ref] [bib_ref] Engineered gene circuits, Hasty [/bib_ref]. There are, however, many cellular processes, such as macromolecular assembly, that cannot naturally be described in terms of chemical reactions. One of the major challenges of current biology is therefore to incorporate the molecular details into the description of the dynamics of cellular processes. We have presented here an approach for bridging the gap between molecular properties and the dynamics of networks of interactions. It can be applied in general to compute the stochastic dynamics of macromolecular assembly networks and their integration into cellular networks. This method is based on a binary description of the potential states of the system and a decomposition of the free energy into a combination of a small subset of elementary contributions of the different components. Such decomposition not only brings forward an extra level of regulation but also provides a starting point to characterize and predict the collective properties of macromolecular complexes, such as looped DNA-protein complexes, in terms of the properties of their constituent elements. The thermodynamic grounds of the method allow for the use of the principle of detailed balance to obtain rate constants, which prevents the appearance of the unrealistic situations noted in existing approaches [bib_ref] Computer-based analysis of the binding steps in protein complex formation, Bray [/bib_ref] [bib_ref] Automatic generation of cellular reaction networks with Moleculizer 1.0, Lok [/bib_ref]. The two examples explored here, the induction switches in the lac operon and in phage l, represent perhaps the most elementary gene regulatory networks of the most basic organisms. And yet, to fully understand the transcriptional regulation in both systems one has to consider thermodynamic quantities that extend beyond standard theory of chemical reactions: macromolecular assembly networks have mecha-nisms built in that can be used to increase specificity and affinity simultaneously and, at the same time, to control the inherent stochasticity of cellular processes. In particular, exploiting the flexibility of DNA [bib_ref] Spontaneous sharp bending of doublestranded DNA, Cloutier [/bib_ref] [bib_ref] Inferring the in vivo looping properties of DNA, Saiz [/bib_ref] , protein-DNA complexes can lead to the suppression of cell-to-cell variability, the control of transcriptional noise, and the activation of cooperative interactions on demand [bib_ref] DNA looping and physical constraints on transcription regulation, Vilar [/bib_ref] [bib_ref] DNA looping in gene regulation: from the assembly of macromolecular complexes to..., Vilar [/bib_ref]. DNAprotein complexes take full advantage of the conformational properties of DNA by introducing long-range interactions, thus making DNA an active participant in the delivery of the information it encodes. The mathematical part of the method we have presented has a direct correspondence with a graphical network description, where nodes represent whether or not an element or a property of the component is present, and where the strength of the links between nodes carries information about the effects of these elements on the stability of the complex. Therefore, our approach accounts for the precise stochastic biochemical dynamics, as shown by the prototype systems considered here, while keeping the simplicity of qualitative methods based on network diagrams. This methodology thus offers a solid starting point to move from qualitative to quantitative understanding of protein-protein [bib_ref] A Bayesian networks approach for predicting protein-protein interactions from genomic data, Jansen [/bib_ref] , protein-DNA [bib_ref] Bridging structural biology and genomics: assessing protein interaction data with known complexes, Edwards [/bib_ref] , and other interaction networks [bib_ref] Systematic determination of genetic network architecture, Tavazoie [/bib_ref] [bib_ref] Network motifs in the transcriptional regulation network of Escherichia coli, Shen-Orr [/bib_ref] on a genomic scale. [fig] Figure 1: Representation and thermodynamics of macromolecular assembly. (A) An example of a macromolecular complex in one of eight possible states is used as illustration of the binary description. The complex consists of three molecular positions A, B, and C, described by binary variables s A , s B , and s C , respectively. In this case, A and C are occupied (gray shapes) and B is unoccupied (white shape with dashed contour). Red lines represent pairwise interactions between the components. This description can easily be connected to the thermodynamic properties of different configurations. Here, the free energy DG o for the configurations and their contributions are expressed in units of kcal/mol. The positional and interaction free energies are assumed to be 15 and À20, respectively. Note that the description refers to a three-molecule complex at a specific location. If, instead, one molecule is used as a reference, its positional free energy should not be counted. The free energy of the disconnected configuration (B) is much higher than the free energy of the connected configuration (C). These energetic considerations indicate that the disconnected configuration is extremely less abundant than the connected one. The stability of a compact structure (D) is considerably higher than that of a chain-like structure (C) because of the additional free energy of interaction. [/fig] [fig] Figure 2: Interaction networks. The state and properties of the macromolecular structure can be described by an interaction network. Nodes (big gray circles) in the interaction network represent whether or not a component is present. Small black circles are joined to nodes and represent interactions between the elements they join. Labels associated with black circles indicate the contributions to the free energy arising when all the nodes they are linked to are occupied. This graphical representation is equivalent to the mathematical expression of the free energy in terms of binary variables. For the network shown here, the free energy of a state s¼(s A ,s B ,s C ) is given by DG(s)¼ p(s A þ s B þ s C ) þ e AB s A s B þ e BC s B s C þ e CA s C s A , where p is the positional free energy and e AB , e BC , and e CA are the interaction free energies between the different components. Instantiating all the possible values of the state variable s leads to eight states, which have a Markov Chain (Norris, 1997) graphical representation in which nodes indicate each specific state s A s B s C of the complex and arrows indicate transitions from one state to another. Only transitions in which a component gets in or out of the complex are displayed here. [/fig] [fig] Figure 3: Repressor-DNA assembly and gene regulation in the lac operon. (A) The lac repressor's binding to DNA can be described by a network of interacting binary elements. Gray circles represent the two DNA-binding domains of the lac repressor bound at the DNA sites O1 (1) and O2 (2) and the lines and black small circles represent interactions. The gray polyhedron represents whether or not DNA is looped (L). DG(s) is the expression of the free energy of the complex in terms of the binary variables. (B) The cartoon illustrates the lac repressor (green) bound to looped DNA (orange), with the circles and the polyhedron indicating the repressoroperator DNA binding sites and the DNA loop, respectively. The binary variables, s 1 and s 2 , are 1 if the corresponding repressor-operator interaction takes place and are 0 otherwise. Contributions to the free energy (in units of kcal/mol) of the complex include positional (p¼15À0.6 ln[N]; considering DG pos o E15 (Finkelstein and Janin, 1989) and RT¼0.6) and repressor-DNA interaction (e 1 ¼À28.1 and e 2 ¼À26.6) terms. Here, [N] is the lac repressor concentration expressed in moles. For the looped DNA complexes (s L ¼1), there are also contributions from the cost of DNA looping (c L ¼23.35) and the interaction between sites 1 and 2 mediated by DNA looping (Àp). [/fig] [fig] Figure 5: Phage l cI self-regulation kinetics. (A) [/fig]

Delocalization of Electrons in Strong Insulators at High Dynamic Pressures Systematics of material responses to shock flows at high dynamic pressures are discussed. Dissipation in shock flows drives structural and electronic transitions or crossovers, such as used to synthesize metallic liquid hydrogen and most probably Al 2 O 3 metallic glass. The term -metal‖ here means electrical conduction in a degenerate system, which occurs by band overlap in degenerate condensed matter, rather than by thermal ionization in a non-degenerate plasma. Since H 2 and probably disordered Al 2 O 3 become poor metals with minimum metallic conductivity (MMC) virtually all insulators with intermediate strengths do so as well under dynamic compression. That is, the magnitude of strength determines the split between thermal energy and disorder, which determines material response. These crossovers occur via a transition from insulators with electrons localized in chemical bonds to poor metals with electron energy bands. For example, radial extents of outermost electrons of Al and O atoms are 7 a 0 and 4 a 0 , respectively, much greater than 1.7 a 0 needed for onset of hybridization at 300 GPa. All such insulators are Mott insulators, provided the term -correlated electrons‖ includes chemical bonds. # Introduction Electrons in strong insulators are delocalized at high pressures. A strong insulator is defined to be one that has a high bulk modulus and a wide band-gap at ambient. A weak insulator is defined here to be one that has a small bulk modulus and a wide band-gap at ambient. Examples are sapphire (single-crystal Al 2 O 3 ) and liquid H 2 , respectively. Their band-gaps at ambient are 10 eV and 15 eV, respectively. Sapphire is one of the most incompressible materials known and condensed H 2 is one OPEN ACCESS of the most compressible materials. Despite the great disparity in strength and compressibility, both of these wide band-gap insulators undergo a continuous transition or crossover to poor metals at ~100 GPa shock pressures and probably by the same general mechanism. The purpose of this paper is to discuss the likely reason why such different insulators would behave so similarly with respect to the onset of electrical conduction with essentially the same value of conductivity-minimum metallic conductivity. ## Basic idea under dynamic pressure An electronic transition to a metallic state is called an insulator-metal transition (IMT). Many IMTs are associated with structural phase transitions or crossovers, as likely occurs in sapphire to an amorphous state at a shock pressure of ~300 GPa (3 million bar = 3 Mbar) [bib_ref] Al 2 O 3 as a metallic glass at 300 GPa, Nellis [/bib_ref] and does occur in liquid H 2 at 140 GPa [bib_ref] Minimum metallic conductivity of fluid hydrogen at 140 GPa (1.4 Mbar), Nellis [/bib_ref]. While one would normally not expect materials with such an extreme difference in strength to have much in common in terms of an IMT, the metallization processes in these two at high shock pressures are probably extreme limits of the same general process. One key to recognizing this situation is the fact that crossovers to poor metals in both fluid hydrogen and amorphous Al 2 O 3 are accomplished by dynamic or shock compression. Shock compression generally means compression by a single shock wave; dynamic compression is a more general term that often means compression by multiple shock waves or by ramp waves in time. In this materials context, the difference is a detail. The important point with respect to materials is that shock and dynamic compression are dissipative and dissipation in shock flows drives atomic disorder and electron delocalization in amorphous solids and in fluids. Shock compression is dissipative, in the sense that not all the energy deposited by shock compression goes into compression; i.e., into PdV work, where P is pressure and V is volume. Dissipation energy goes into temperature T, or non-equilibrated thermal energy, and entropy S, thermally equilibrated or not. Entropy is a word used here to denote the concept of damage or disorder. Shock-induced IMTs have been observed at elevated shock temperatures (T ≥ 1,000 K) and are thus often called semiconductor-metal transitions (SCMT). The basic idea is that at sufficiently high dynamic pressure there is sufficient dissipation in the form of thermal energy and entropy to destroy chemical bonds, which localize electrons at ambient. Once chemical bonds are destroyed with high dynamic pressures, wave functions of disordered atoms overlap at sufficiently high densities for itinerant energy bands to form. In Al 2 O 3 and H 2 the bonds are Al-O and H-H and both bonds have the same bond strength, 4.5 eV. On the other hand, their material structures are totally different. At low pressures Al 2 O 3 has a rigid, three-dimensional (3D), ordered crystal structure with a strong bond between all Al-O pairs in the 3D lattice. In contrast liquid H 2 is a disordered structure in which H 2 molecules interact via a weak Vander Waals pair potential with a well-depth of 0.004 eV [bib_ref] The equation of state of molecular hydrogen at very high density, Ross [/bib_ref]. The different initial structures significantly affect the way these two materials respond on a microstructural scale to dynamic pressure. However, in both cases chemical bonds eventually dissociate under sufficiently high dynamic pressure. Once the bonds are destroyed, electrons that were correlated in local chemical bonds have spherically symmetric or spherically-averaged atomic wave functions that hybridize into itinerant energy bands of a metal at sufficiently high densities. Shock pressures in the 100 GPa range produce sufficiently high densities. This process is termed Bonds to Bands (BtB). Material strength determines the split between thermal energy and entropy, but not whether or not a BtB can occur. Because of the highly disordered metallic state, conduction electron scattering is strong and so -metallic conductivity‖ means minimum metallic conductivity (MMC ≈ 2,000/(Ω-cm) = 500 µΩ-cm.), as observed in fluid H 2 [bib_ref] Minimum metallic conductivity of fluid hydrogen at 140 GPa (1.4 Mbar), Nellis [/bib_ref] and as predicted based on modest extrapolation of measured electrical conductivities of Al 2 O 3 shock-compressed up 220 GPa [bib_ref] Al 2 O 3 as a metallic glass at 300 GPa, Nellis [/bib_ref]. ## Possibilities under static pressure Several strong insulators also probably reach MMC in a diamond-anvil cell (DAC) at 100 GPa static pressures at 300 K. These are ones with frustrated phase transitions that produce amorphous samples at sufficiently high static pressures. An example is Al 2 O 3 , which has been compressed in a DAC up to 200 GPa [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref]. In the as-compressed state, only a disordered α-corundum phase was observed up to 180 GPa in a DAC. Laser heating up to ~2,000 K was required at high pressures in order to drive and then thermally quench high-pressure phases to ambient temperature at high pressure. For the Al 2 O 3 sample compressed to 200 GPa, no crystalline symmetry was reported, which presumably means the Al 2 O 3 sample compressed statically to 200 GPa was amorphous. It is possible that amorphous Al 2 O 3 reaches MMC at ~300 GPa in a DAC, as suggested previously [bib_ref] Al 2 O 3 as a metallic glass at 300 GPa, Nellis [/bib_ref]. A similar situation occurs in Gd 3 Ga 5 O 12 (GGG) [bib_ref] High-pressure and high-temperature studies on oxide garnets, Hua [/bib_ref] [bib_ref] Transition to a virtually incompressible oxide phase at a shock pressure of..., Mashimo [/bib_ref] [bib_ref] Equation of state of a high-pressure phase of Gd 3 Ga 5..., Mao [/bib_ref] , as discussed below. ## Mott-like insulators The notion that chemical bonds in electrical insulators localize electrons and that electron delocalization is achieved by breaking those bonds to form metallic energy bands is similar to the idea of a Mott insulator, of which NiO is a paradigm [bib_ref] The basis of the electron theory of metals, with special reference to..., Mott [/bib_ref]. Based on its band structure, NiO is expected to be a metal. However, it is actually a transparent, anti-ferromagnetic insulator. Mott proposed that many-body electron correlations localize electrons at ambient, and that an IMT probably occurs at high pressures. A chemical bond consists of correlated-electron wave functions and their associated correlated electron spins. The cases considered here involve nonmagnetic insulators, which traditionally have not been considered to be Mott insulators. To distinguish classical magnetic Mott insulators from ones with shock-induced SCMTs in nonmagnetic crystals, I call the latter ones Mott-like. However, Mott and Mott-like insulators are essentially the same, provided the definition of Mott insulator is generalized to include all insulators with electron correlations that localize electrons at ambient, magnetic or not, and become metallic at high pressures. ## Dynamic compression Extreme states of condensed matter are generated via the coupling between shock hydrodynamics and either equilibrium thermodynamics or non-equilibrium microstructures. Pressures used in experiments discussed herein were single-shock pressures in the case of Al 2 O 3 and multiple-shock compression in the case of H 2 . Shock pressures were generated by high-velocity (1 to 8 km/s) impacts of a planar solid disc onto a sample target at rest. Experimental lifetimes were typically ~100 ns. Rise times of shock pressures ranged from a few tenths to a few tens of ns. Because of the fast time scale, the method is called dynamic compression. Because of the 100 ns lifetime, dynamic compression is adiabatic (too brief for heat to diffuse out of a sample), too brief for highly-mobile shock-compressed and heated hydrogen to diffuse out of its sample holder, sufficiently long for fluids and metals to equilibrate thermally, and too brief for strongly covalently-bonded materials to equilibrate thermally. A general discussion of dynamic compression has been published [bib_ref] Dynamic compression of materials: Metallization of fluid hydrogen at high pressures, Nellis [/bib_ref]. In this paper we present arguments for the fact that metallization of all insulators at high dynamic pressures probably occurs via a single general mechanism. Strong and weak insulators, such as sapphire and liquid H 2 , are two extreme cases of this general mechanism and these extreme cases facilitated its recognition. In Section 4 the classical IMT in H 2 is discussed. In Section 5 the SCMT in fluid hydrogen is discussed because it is the first experimental observation of a metallic phase of highly condensed hydrogen, is a reference point for discussion of metallization of strong insulators, and as an illustration of how shock dissipation causes the Bonds-to-Bands Transition (BtBT) in H 2 . It is shock dissipation that enabled metallic fluid H to exist at pressures sufficiently low to be observed in a laboratory. The IMT from solid H 2 to metallic hydrogen at low temperatures (T < 300 K) is yet to be observed experimentally. In Section 6 general shock synthesis of amorphous metallic oxides is discussed. Experimental results for Al 2 O 3 and Gd 3 Ga 5 O 12 , are reviewed, which includes comparing radial extents of electron charge-densities of the various atoms to the size of the average volumes into which they must fit at high pressures. The latter provides a basis for determining if a material is likely to be metallic at a given compression. Basic conclusions are given in Section 7. ## Classical imt in h 2 The IMT from H 2 to H at T = 0 K was proposed by Wigner and Huntington (WH) in 1935 [bib_ref] On the possibility of a metallic modification of hydrogen, Wigner [/bib_ref] and remains today the paradigm of a pressure-driven first-order phase transition. In particular, WH demonstrates the intrinsic importance and simplicity of the dissociative phase transition for achieving a metallic state. The classical IMT of WH was a paradigm for understanding the crossover under dynamic compression from insulating liquid H 2 to metallic fluid H [bib_ref] Minimum metallic conductivity of fluid hydrogen at 140 GPa (1.4 Mbar), Nellis [/bib_ref] , which in turn provided a basis for understanding the likely crossover from strong insulators to disordered poor metals [bib_ref] Al 2 O 3 as a metallic glass at 300 GPa, Nellis [/bib_ref]. So as background for discussions below, we begin with a discussion of the WH IMT in H 2 . WH's classical view of an IMT under pressure is one of a crystalline insulator compressed hydrostatically at temperature T = 0 K and at some high pressure a first-order dissociative phase transition to a metallic state occurs [bib_ref] On the possibility of a metallic modification of hydrogen, Wigner [/bib_ref]. H 2 is an insulator because two electrons are localized in each intramolecular H-H bond. However, on dissociation to H one electron per atom at sufficiently high density means one electron per energy band, which is a band structure of a metal. The dissociative phase transition from H 2 to metallic H occurs simply because of dissociation and the resulting half-filled energy band, independent of band shape. In 1935 WH predicted theoretically the classical IMT in H 2 would occur at an estimated pressure of ~25 GPa. The IMT in solid H 2 has yet to be observed experimentally under static pressures up to ~300 GPa. Optical studies on solid H 2 in a DAC suggest metallic H 2 should be observed at about 450 GPa near 100 K [bib_ref] Optical studies of solid hydrogen to 320 GPa and evidence for black..., Loubeyre [/bib_ref]. The current theoretical estimate of pressure for the dissociative transition from solid H 2 to solid metallic H at T = 0 K is 500 GPa [bib_ref] Ground-state structures of atomic metallic hydrogen, Mcmahon [/bib_ref]. Both of these predicted pressures are well beyond current capabilities of a diamond anvil cell (DAC). It is theoretically possible, in principal, for H 2 to metalize at T = 0 K if its energy gap closes to k B T = 0, where k B is Boltzmann's constant. However, H 2 dissociation energy and band gap near ambient are 4.5 eVand 15 eV [bib_ref] Fundamental absorption spectra of solid hydrogen, Inoue [/bib_ref] , respectively. While the density-dependences of these parameters are not yet known exactly, the fact that the band gap at ambient is a factor of 3.3 times larger than dissociation energy, implies that dissociation at T = 0 K, and thus metallization, is more likely to occur at a lower density/pressure than band-gap closure of H 2 . Thus, the IMT in H 2 probably occurs by dissociation, though this point is yet to be answered by experiment. One likely reason the IMT of WH is yet to be observed is the large H-H bond strength of 4.5 eV, which is also the strength of the Al-O bond. To date it has not been possible to deposit 4.5 eV into solid H 2 to dissociate it by compression alone at T = 0 K. ## Shock dissipation in liquid h 2 : metallic fluid h Since predicted pressures required to metalize hydrogen in a DAC at low temperatures are beyond current DAC capability, it was appropriate to look for the IMT in H 2 by trying something in addition to pure compression, something that might induce the IMT in H 2 at pressures that can be achieved in a laboratory. Heating compressed H 2 in a DAC is the obvious choice because heating might drive dissociation, which produces metal. However, if H 2 at ~100 GPa in a DAC is heated above ~300 K, H 2 diffuses out of a DAC in a few minutes, which thus far has been a major impediment to making metallic hydrogen in a DAC. So the issue then becomes the more complex one of finding a method at finite T. The free energy F of a system is F = U−TS, where U is internal energy and dissipation energy is E d = TS. The method used would need to compress condensed hydrogen adiabatically to a temperature T with a ~100 GPa pressure pulse to increase its density by a factor of ~10 in an experimental lifetime such that hydrogen has insufficient time to diffuse out of the sample holder but sufficient time to thermally equilibrate to T and S. In H 2, dissociation is the dominant contribution to ΔS. Because the method must be adiabatic, pressurization, compression, and heating must be simultaneous. Irreversible shock energy deposited in the multiple-shock compression process is divided between T and S such that their product is maximized in order to minimize the free energy. Thus, at sufficiently large T > 0 and entropy increase ΔS > 0, the SCMT in H 2 might be achieved to a metallic H phase at lower pressures than can be achieved at T = 0 K at the same density. Dynamic compression achieves ~100 GPa pressures, up to 10-fold compression of initial density of liquid H 2 , at temperatures up to ~3,000 K simultaneously for experimental lifetimes of ~100 ns. Because of the fast rise time of pressure (~10 ns) and the short experimental lifetime, dynamic compression is dissipative and adiabatic, respectively. That is, experimental lifetime is too short for heat and hydrogen to diffuse out of the compressed hydrogen sample. Shock dissipation energy E d goes into T and S. This SCMT has in fact been observed experimentally. By simultaneously pressurizing, compressing, and heating liquid H 2 initially at 20 K with multiple-shock compression, MMC of liquid H is achieved at 140 GPa, 9-fold compressed initial liquid-H 2 density, and ~3,000 K [bib_ref] Minimum metallic conductivity of fluid hydrogen at 140 GPa (1.4 Mbar), Nellis [/bib_ref] [bib_ref] Metallization of fluid molecular hydrogen at 140 GPa (1.4 Mbar), Weir [/bib_ref]. Ross has shown this SCMT is facilitated by the fact that H 2 dissociation energy decreases with compression [bib_ref] Temperature measurements and dissociation of shock-compressed liquid deuterium and hydrogen, Holmes [/bib_ref]. At the same time temperature increases with dynamic compression. Thus, at a sufficiently high dynamic pressure, H 2 molecules dissociate to metallic fluid H in a crossover region. The H temperature of 3,000 K is much higher than melting temperatures of H 2 , measured experimentally and calculated theoretically at 140 GPa [bib_ref] Raman spectroscopy of hot dense hydrogen, Gregoryanz [/bib_ref] [bib_ref] A quantum fluid of metallic hydrogen suggested by first-principles calculations, Bonev [/bib_ref] [bib_ref] Melting line of hydrogen at high pressures, Deemyad [/bib_ref] [bib_ref] Evidence of maximum in the melting curve of hydrogen at megabar pressures, Eremets [/bib_ref] [bib_ref] Evidence for a first-order liquid-liquid transition in high-pressure hydrogen from ab initio..., Morales [/bib_ref]. Thus, dense metallic H is a fluid. Because of the large compression, the Fermi temperature T F exceeds 200,000 K, T/T F ~ 0.01, and metallic fluid H is highly degenerate. The highest measured value of electrical conductivity in the fluid is 2,000/(Ω-cm) at pressures from 140 to 180 GPa, which is MMC and consistent with theory of dense hydrogen [bib_ref] Electrical conductivity of high-pressure liquid hydrogen by Quantum Monte Carlo methods, Lin [/bib_ref] [bib_ref] A note on the metallization of compressed liquid hydrogen, Tamblyn [/bib_ref]. Dissociation of the H-H bond achieves spherically symmetric H atoms. Sufficient pressure was available in those experiments to achieve sufficient overlap of electronic wave functions on adjacent H atoms to form an itinerant energy band of a metal. N 2 and O 2 undergo a similar crossover from a diatomic insulator to monatomic metallic fluid. Fluid H, N, and O have similar measured values of MMC (2,000 (Ω-cm) −1 ) at highest pressures (100 to 140 GPa) [bib_ref] Minimum metallic conductivity of fluid hydrogen at 140 GPa (1.4 Mbar), Nellis [/bib_ref] [bib_ref] Metallization of fluid nitrogen and the Mott transition in highly compressed low-Z..., Chau [/bib_ref] [bib_ref] High-pressure insulator-metal transition in molecular fluid oxygen, Bastea [/bib_ref] [bib_ref] Pressure-produced ionization on nonideal plasma in a megabar range of dynamic pressures, Fortov [/bib_ref]. Because T is finite, at dynamic pressures lower than required to achieve MMC, hydrogen, nitrogen, and oxygen are semiconductors. Expanded-fluid Cs and Rb near their liquid-vapor coexistence curves at ~2,000 K reach MMC at ~10 MPa (100 bar) static pressures [bib_ref] The changing phase of liquid metals, Hensel [/bib_ref]. Thus, five elemental monatomic fluids, H, N, O, Rb, and Cs, undergo a Mott-like SCMT under pressure. is a plot of electrical conductivities of H, N, O, Rb, and Cs versus a*/D 1/3 , the ratio of atom size, assumed to be the effective Bohr radius a*, to the average distance between adjacent atoms in the fluid, which is determined by density and thus by pressure. D is the volume of the average cube around each atom. D 1/3 is average distance between adjacent nuclei. Plotting data this way for systems near an IMT was suggested by Mott, and applied by Hensel to his Rb and Cs conductivity data [bib_ref] The changing phase of liquid metals, Hensel [/bib_ref]. implies the radial extents of wave functions of Rb and Cs are relatively large because relatively little compression of Rb and Cs is required for them to conduct. Similarly, H requires substantial compression in order to conduct. N and O are intermediate but closer to H. also shows that as atoms are pushed together by pressure, conductivity increases until at highest pressures all five elements have MMC (2,000 (Ω-cm) −1 ). Since a*/D 1/3~0 .35-0.38 for 5 elements at MMC, overlap of wave functions on adjacent atoms is substantial for all of them. The maximum value a*/D 1/3 can have is 0.5, which corresponds to coincidence of maxima in electron densities on adjacent atoms. The idea that large overlap is also probably required for oxides to become poor metals at high pressures arises below in the discussion of Al 2 O 3 and Gd 3 Ga 5 O 12 . The trend above is as expected. For example, attraction by the Cs nucleus of the outer 6s 1 conduction electron is screened by a Xe core. Substantial screening means the radial extent of the 6s 1 electron is relatively large and so relatively little compression is needed to obtain sufficient overlap of wave functions on adjacent sites to cause the onset of electrical conduction. For H, the opposite is true. The attraction between electron and proton is unscreened and so substantial compression is expected before the onset of conduction in fluid H. Screening in N and O is relatively small compared to that in Cs. That is, screening in N and O is caused by filled 1s 2 and 2s 2 electron shells, which are relatively small compared to the size of a Xe core in Cs. Thus, onset density of conductivity in N and O is closer to that in H. Statements about expected radial extents of atomic wave functions derived from are readily checked by comparing radial electron-density distributions calculated in the Hartree-Fock-Slater approximation for the five elements [bib_ref] Metallization of fluid nitrogen and the Mott transition in highly compressed low-Z..., Chau [/bib_ref]. These results are shown in . Inspection of (b) shows that the radial extent of the electron density distribution for H is least, greatest for Rb and Cs, and intermediate for N and O as well as being closer to that of H than to that of Rb and Cs, as expected. ## Figure 1. Electrical conductivities of H, N, O, Rb, and Cs plotted versus a*/D 1/3 , where a* is size of atom (effective Bohr radius) and D is volume of average cube around each atom. D 1/3 is average distance between adjacent nuclei. Each point is measured electrical conductivity at specific pressure [bib_ref] Metallization of fluid nitrogen and the Mott transition in highly compressed low-Z..., Chau [/bib_ref]. ## Figure 2. Electron densities (4πr 2 ψ*ψ) plotted versus radii [bib_ref] Metallization of fluid nitrogen and the Mott transition in highly compressed low-Z..., Chau [/bib_ref]. To look at radial extents of outer electrons, for comparison purposes peak of each distribution was shifted to r = 1 bohr in plots. Curves in (b) are curves in (a) plotted on expanded scale. Fluid H, N, O, Rb, and Cs have been discussed in some detail to demonstrate that Mott plots of measured electrical conductivities versus Mott parameter a*/D 1/3 provide qualitative estimates of the relative radial extents of electron density distributions, calculated in the Hartree-Fock-Slater approximation. This extensive self-consistency between experimental results and atomic structure calculations has two significant implications. First, it is reasonable to use radial electron-density distributions of atoms calculated in the Hartree-Fock-Slater approximation to make estimates about densities at which materials more complex than simple fluids might be metallic at extreme conditions. In this regard, likely metallization pressures and densities of Al 2 O 3 and Gd 3 Ga 5 O 12 are discussed in the next section. Second, electrical conductivities of elemental fluids measured with a 20-m long two-stage light-gas gun enable resolution of radial extents of quantum mechanical electron density distributions on the spatial scale of ~Bohr. If metastable solid metallic hydrogen (MSMH) could be quenched to ambient pressure and temperature, this material might have several scientific and technological uses including: a quantum solid with novel physical properties, including room-temperature superconductivity; a very lightweight structural material; a chemical fuel, propellant, or explosive, depending on the rate of release of stored energy; a dense nuclear-fusion fuel made with isotopes deuterium and tritium, rather than hydrogen, to obtain higher energy yields in inertial confinement fusion [bib_ref] Metastable solid metallic hydrogen, Nellis [/bib_ref]. In summary, under dynamic compression, -soft‖ fluids, such as H 2 rapidly undergo large compressions, which induce high dynamic temperatures. In such systems thermal equilibrium is generally achieved in a sub-ns time scale. As dynamic pressure increases H 2 dissociates which causes entropy to increase and thermally equilibrate also on a sub-ns time scale. Thus, dynamically compressed fluids can be treated with equilibrium thermodynamics. In this case relatively simple assumptions and approximations give theoretical results in generally good agreement with experiment. Representative examples are Ross' work on dissociation of H 2 [bib_ref] Temperature measurements and dissociation of shock-compressed liquid deuterium and hydrogen, Holmes [/bib_ref] and Mott's MMC. ## Shock dissipation in strong insulators: likely synthesis of metallic oxide glasses Strong insulators differ substantially in most respects from weak fluid insulators discussed in Section 5. Interaction potentials differ substantially, breaking strong bonds in a rigid 3D lattice requires several eV, and atom densities in strong insulators near ambient are quite high compared to fluids. As a result, up to 10-100 GPa shock pressures, shock dissipation in dense strong insulators is absorbed substantially by mechanically breaking and bending inter-atomic bonds. In fact in this range of shock pressures, shock-induced damage and heating are often heterogeneous and T and S do not equilibrate thermally in bulk during experimental lifetimes until shock pressures exceed ~100 GPa. Nevertheless, with modest extrapolations of existing conductivity data of Al 2 O 3 and Gd 3 Ga 5 O 12 up to ~250 GPa, both likely reach MMC at 300-400 GPa, as do compressible fluid H, N, and O at ~100 GPa. ## Al 2 o 3 (sapphire) Sapphire with a density of 3.98 g/cm 3 disorders substantially under shock pressures up to ~100 GPa [bib_ref] Entropy-dominated dissipation in sapphire shock-compressed up to 400 GPa (4 Mbar), Nellis [/bib_ref] and references therein). It is likely that shock heating of Al 2 O 3 is not uniform in bulk until shock pressures exceed ~200 GPa. Moreover, the Hugoniot and 300-K isotherm of sapphire are nearly coincident up to 400 GPa. Above ~400 GPa, Hugoniot pressure increases dramatically and diverges from the 300-K isotherm. These observations suggest a picture in which entropy dominates dissipation below ~400 GPa and once entropy is maximized total pressure and thus shock temperature and thermal pressure increase rapidly with additional shock compression. In a DAC, the corundum (α-Al 2 O 3 ) to Rh 2 O 3 (II)-type phase transition occurs at 103 GPa and the Rh 2 O 3 (II)-type to CaIrO 3 -type transition occurs at 130 GPa and persists to at least 180 GPa. However, these transitions in a DAC are sluggish. They occur only with laser-heating and thermal quenching at high pressures. X-ray spectra of laser-heated and quenched samples consist of broad individual diffraction peaks superimposed on a significant broad background, indicative of disordered structures with short-range order [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref]. Those samples in a DAC are substantially disordered, which means a substantial amount of entropy. The effect of shock-induced disorder on measured electrical resistivities of sapphire at shock pressure from 91-220 GPa [bib_ref] Electrical resistivity of single-crystal Al 2 O 3 shock-compressed in the pressure..., Weir [/bib_ref] is illustrated in [fig_ref] Figure 3: Measured electrical resistivity of sapphire plotted versus shock pressure up to 220... [/fig_ref]. Up to ~130 GPa electrical resistivity is large and essentially constant. From 130-220 GPa, resistivity decreases by a factor of 10 3 and extrapolates to 500 µΩ-cm around 280 GPa. 500 µΩ-cm corresponds to electrical conductivity of 2,000 (Ω-cm) −1 , which is MMC, the same MMC as metallic fluid H reaches at 140 GPa, 9-fold compressed liquid-H 2 density, and ~3,000 K (Section 5). In sapphire a static pressure of 130 GPa is the pressure of the Rh 2 O 3 (II)-type to CaIrO 3 -type transition in a DAC, and CaIrO 3 -type disorders increasingly at higher pressures in a DAC. The picture that emerges from all these experiments is that shocked sapphire damages in the corundum (α-Al 2 O 3 ) and Rh 2 O 3 (II)-type phases; from 130-280 GPa shocked sapphire undergoes a crossover with increasing disordering. At ~280 GPa shocked sapphire has MMC, which suggests it is an amorphous atomic metal or metallic glass, basically a frozen fluid. Of course, the possibility exists that sapphire is a fluid at ~280 GPa because the melting curve of sapphire has yet to be measured at these pressures. Alternatively, Al and O might phase separate at these extreme conditions, which might mean that current is conducted through Al filaments surrounded by non-conductive oxygen. This is conceivable because Al composition is 40 at.% which exceeds the percolation limit for filamentary conduction. A key issue to consider is whether or not it is possible for Al and O to form hybridized energy bands and, thus, have a band structure of a disordered Al-O metallic alloy. Chemical bonds have a characteristic length and energy. Shock compression is about a factor of 1.6 in density, which would shorten and distort bond lengths. Shock dissipation supplies energy to substantially disorder strong insulators. So it is reasonable to assume that all bonds are broken at sufficiently high shock pressures, which produces a disordered collection of Al and O atoms whose wave functions might hybridize. At shock compression of 1.6 in density and under the assumption that both Al and O atoms must fit into an average-size cube with the same volume, then each atom must fit into a cube with an edge length of 3.3 a 0 , where a 0 is the Bohr radius. Thus, if the radial extents of the electron density distributions of Al and O atoms exceed 1.7 a 0 , then wave functions on adjacent atoms will overlap and it will be possible for them to hybridize in the band structure of a disordered solid solution. Radial extents of the outermost electrons of Al (3s 2 3p 1 ) and O (2p 4 ) atoms were calculated in the Hartree-Fock-Slater approximation [bib_ref] Al 2 O 3 as a metallic glass at 300 GPa, Nellis [/bib_ref] and are shown in [fig_ref] Figure 4: Spherically averaged atomic electron densities versus radius for O 2p 4 and... [/fig_ref]. The radial extents of outermost electrons of Al and O atoms are 7 a 0 and 4 a 0 , respectively, both of which are much greater than 1.7 a 0 needed for onset of hybridization. Overlap would be substantial, as in the case of compressible fluids discussed in Section 5. Thus, it is spatially possible for Al and O to form hybridized energy bands of a metal in such a dense atomic glass. Thus it is possible that MMC at ~280 GPa in [fig_ref] Figure 3: Measured electrical resistivity of sapphire plotted versus shock pressure up to 220... [/fig_ref] is caused by hybridization of Al and O wave functions and strong electron scattering in an amorphous metallic alloy., DAC data on 300-K isotherm [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref] , and theory [bib_ref] Prediction of an U 2 S 3 -type polymorph of Al 2..., Umemoto [/bib_ref] for Al 2 O 3 up to 340 GPa. Static compression to all data points shown produced only disordered corundum [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref]. Laser-heating and thermal quenching at high pressures to 300 K were required to obtain those data points in [fig_ref] Figure 5: compares the Hugoniot [/fig_ref]. Static compression of Al 2 O 3 without a pressure medium and without laser-heating might produce a sample in a DAC with similar disorder as states on the Hugoniot at comparable compression. In this way it might be possible to synthesize an amorphous Al 2 O 3 sample in a DAC for characterization. An amorphous Al 2 O 3 sample in a DAC might have MMC at ~300 GPa, as well as under shock compression., DAC data on 300-K isotherm [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref] , and theory [bib_ref] Prediction of an U 2 S 3 -type polymorph of Al 2..., Umemoto [/bib_ref] for Al 2 O 3 up to 340 GPa. ## Gd 3 ga 5 o 12 (ggg) GGG, with a density of 7.10 g/cm 3 and no long-range magnetic order, disorders substantially under static and shock compression. In a DAC, GGG is crystalline up to 74 GPa, above which x-ray diffraction peaks broaden up to 84 GPa, above which GGG is amorphous [bib_ref] High-pressure and high-temperature studies on oxide garnets, Hua [/bib_ref]. Hugoniot data of GGG single crystals have been measured from 30 to 260 GPa. The Hugoniot elastic limit (HEL) of GGG was found to be 30 GPa, a transition to an intermediate (IM) phase occurs at 65 GPa and extends up to 120 GPa, followed by the onset of a virtually incompressible high-pressure (HP) phase that extends up to 260 GPa. Calculated shock temperatures of GGG reach 6500 K at 260 GPa. Up to ~70 GPa the pressure-volume data measured in a DAC at 300 K and the Hugoniot are virtually identical. From shock pressures of 120 up to 260 GPa (HP phase), electrical conductivity measurements show that GGG is semiconducting [bib_ref] Transition to a virtually incompressible oxide phase at a shock pressure of..., Mashimo [/bib_ref]. The sharp increase in slope of shock pressure versus compression of GGG at ~120 GPa and ~1,000 K [bib_ref] Transition to a virtually incompressible oxide phase at a shock pressure of..., Mashimo [/bib_ref] could be caused by a transition to a virtually incompressible phase (a density effect) or by onset of substantial shock temperature and thus thermal and total pressure. Mao et al. found that GGG becomes amorphous in a DAC at 88 GPa and transforms to a new high-pressure phase at 88 GPa on laser heating to 1500 K [bib_ref] Equation of state of a high-pressure phase of Gd 3 Ga 5..., Mao [/bib_ref]. The high-pressure phase in a DAC is cubic, consistent with a perovskite structure and stoichiometry of (Gd 0.75 Ga 0.25 )GaO 3 , and persists up to 180 GPa at 1,500 K. No rapid increase in pressure with compression is observed up to 180 GPa in the DAC data. The DAC data imply the rapid increase in shock pressure with compression at 120 GPa is caused by a combination of fast incomplete phase transitions and increasing shock heating, which is consistent with observed semiconductivity. It is interesting to note that Al 2 O 3 , as well as GGG, must also be laser heated at 100 GPa pressures in a DAC to induce high-pressure crystalline phases [bib_ref] Stability and compressibility of the high-pressure phases of Al 2 O 3..., Ono [/bib_ref]. Time-resolved shock-wave profiles and Hugoniot data of single-crystal GGG have also been measured from 8.5 to 113 GPa [bib_ref] Pressure-dependent Hugoniot elastic limit of Gd 3 Ga 5 O 12 single..., Zhou [/bib_ref]. The HEL increases from 8 to 24 GPa as final shock pressure increases from 8.5 to 89 GPa. Such a strong pressure dependence of the HEL has not been reported previously and suggests metastability and disorder during experimental lifetimes at those pressures. The phase transition observed at 76 GPa is probably the one observed by Mashimo et al. at somewhat lower pressure. It is interesting to note that single-crystal Al 2 O 3 also has very unusual shock-wave profiles as GGG at comparable shock pressures, which are also indicative of disorder [bib_ref] Response of seven crystallographic orientations of sapphire crystals to shock stresses of..., Kanel [/bib_ref]. Extrapolation of electrical conductivities of GGG measured up to 260 GPa suggest GGG reaches MMC at ~400 GPa (0.4 TPa). In order to determine if this metallization is reasonable, once again the radial extents of atomic charge densities were compared to the size of the average cube into which each must fit at pressure. The highest pressure phase of GGG in a DAC is cubic with an effective stoichiometry of (Gd 0.75 Ga 0.25 )GaO 3 . As for Al 2 O 3 , radial extents of atomic charge densities of outermost electrons of Gd (5d 1 6s 2 ) and Ga (4s 2 4p 1 ) were calculated with the Hartree-Fock-Slater method. Atomic Gd has a radial charge density distribution that is more extended with a lower broad maximum than that of Ga, which is very similar to that of Al. We assume that at 0.4 TPa and compression of 1.9 over initial crystal density, bonds in crystalline Gd 3 Ga 5 O 12 are broken in a glass and Gd, Ga and O atoms can be treated as spherically averaged and symmetric. Calculated radial extents of 5d 1 6s 2 , 4s 1 4p 2 , and 2p 4 electrons of Gd, Ga and O atoms are 8 a 0 , 7 a 0 , and 4 a 0 , respectively. These wave functions must fit into an average cube with an edge length of 3.5 a 0 , which means the distance from the center of the cube to a face is 1.8 a 0 , which is substantially less than the radial extents of outer electrons Gd, Ga, and O. Thus, substantial overlap of atomic wave functions occurs at 0.4 TPa in GGG and it too is likely a metal by band overlap at these extreme conditions. # Conclusions Dissipation in shock flows at high dynamic pressures drives structural and electronic transitions or crossovers, such as to metallic liquid hydrogen and most probably Al 2 O 3 and Gd 3 Ga 5 O 12 metallic glasses. These crossovers occur via Mott-like transitions from chemical bonds in which electrons are localized to disordered metallic systems in which electrons hybridize into itinerant energy bands and have MMC by virtue of strong electron scattering. Dissipation is energy that does not go into compression at pressure-thermal energy and disorder or temperature T and entropy S if in thermal equilibrium. Contrary to popular opinion, dissipation is more than shock heating and significant shock heating is not required to make a poor metal. The term -metal‖ here means electrical conduction in a degenerate system, which occurs by band overlap, rather than thermal ionization. Since H 2 and probably disordered Al 2 O 3 become poor metals with MMC, virtually all insulators with intermediate strengths probably do so as well. [fig] Figure 3: Measured electrical resistivity of sapphire plotted versus shock pressure up to 220 GPa[32]. [/fig] [fig] Figure 4: Spherically averaged atomic electron densities versus radius for O 2p 4 and Al 3s 2 3p 1 electrons calculated with Hartree-Fock-Slater method[1,29]. [/fig] [fig] Figure 5: compares the Hugoniot [/fig]

Role of Intelligent Management Systems in Surgical Punctuality and Quality of Care Objective. e main objective is to illustrate the role of intelligent management systems in surgical punctuality and quality of care. Methods. 72 registered nurses were selected from our operating room, and 180 patients who needed surgery were randomly divided into the control group and the observation group for satisfaction survey and satisfaction analysis. Results. e correct rate of surgical clothing distribution and the qualified rate of clothing recovery were improved, and the punctuality rate of the operation was enhanced than before the implementation of the intelligent management system. e accurate positioning of surgical items and the accurate statistics of equipment use time were enhanced than before implementation. e error rate of surgical item preparation after implementation was lessened than before implementation. Both nursing satisfaction and patient satisfaction after implementation were increased than before implementation. Conclusion. e intelligent management system improves the punctuality of surgery and the quality of care in the operating room. # Introduction Operation room is usually involved in multiple departments which offer important treatment and rescue for patients. e patients admitted to the operation room are generally with serious diseases and complex situations, which require the high-quality nurses. Nurses play an essential role in the operating room work, and the health and safety of patients are closely related to the effectiveness of nursing in the operating room. Traditional operating room management mostly adopted manual recording which not only increases the work burden but also prone to mistakes. In addition, the risk of adverse events easily occurred due to the complexity of operating room nursing work. Strengthening nursing quality in the management of the operating room and standardizing the behavior and operation of nursing staff contribute to reducing the incidence of adverse events and improving the satisfaction rate of patients. With the development of medical technology and the improvement of surgical quality requirements, the hospital management staff turning attaches importance to the master of operating room nursing technology to focus on the exploration of scientific management modes [bib_ref] Clinical decision support systems for triage in the emergency department using intelligent..., Fernandes [/bib_ref] [bib_ref] Zhongguo yi liao qi xie za zhi, Chen [/bib_ref]. Recently, the establishment of intelligent information in medical and health institutions throughout the country provides great convenience for medical staff. Moreover, the work efficiency of medical staff can be effectively improved based on the internet of intelligence and security [bib_ref] IoT operating system based fuzzy inference system for home energy management system..., Ain [/bib_ref]. As a new management mode, an intelligent management system has been employed to operating room work. Intelligent operating room refers to the application of modern information technology in the operating room, and the use of high-tech software and hardware facilities can achieve modern operation process management [bib_ref] Operating room data management: improving efficiency and safety in a surgical block, Agnoletti [/bib_ref]. e entry and exit of operating room medical staff can be obviously controlled by utilizing an information management system, which greatly reduces the wear of operating clothes and the operating cost of the hospital [bib_ref] Emotional intelligence in the operating room: analysis from the Boston Marathon bombing, Chang Md [/bib_ref]. Moreover, the integration of all data in the operating room can significantly control the arrival time of medical staff in the operating room and improve the on-time opening rate [bib_ref] Multi-perspective workflow modeling for online surgical situation models, Franke [/bib_ref]. rough the management of medical practices and transportation of medical supplies, the patient's intraoperative and postoperative conditions can be followed up in time, which is convenient for medical staff to adjust the treatment plan according to the different states of patients [bib_ref] Real-time streaming of surgery performance and intraoperative imaging data in the hybrid..., Lin [/bib_ref]. Comprehensive operating room multisystem information can achieve information integration, effectively manage the flow of people, logistics, and highly improve the operation efficiency and perioperative quality control management. In the present study, the improvement effect of an intelligent process management system on work quality in the digital process of the operating room was explored and analyzed the changes in related work quality of medical staff and patients' job satisfaction before and after implementation. # Methods ## Participants. e study was approved by the ethics committee of Huashan Hospital, which is affiliated with Fudan University in Shanghai. From June 2019 to September 2021, 72 registered nurses aged 22 to 55 years old with an average age of (35.69 ± 8.456) were selected from the operating room of our hospital. 17122 surgeries were collected and adopted the traditional management mode and the intelligent management system in the operating room, respectively. 180 surgical patients ranging from 20 to 70 years old with average ages of (47.68 ± 12.76) years old and (44.97 ± 11.48) years old were randomly divided into prior implementation group (control group) and after implementation group (observation group) to investigate for satisfaction. e control group included 53 males and 37 females, and the observation group included 57 males and 33 females. ere was no statistical divergence in baseline data between the two groups. ## Inclusion and exclusion Criteria. Inclusion criteria were as follows: I) all nurses have professional qualification certificate; II) the nursing age was more than 1 year and had working experience in a general hospital; III) the nurses are all women; IV) the patients requiring surgery; V) all participants signed informed consent. Exclusion criteria were as follows: I) further education nurses; II) the nursing age was lower than 1 year; III) nurses without working experience in general hospital; IV) patients who do not require surgery; V) patients who do not sign informed consent. ## Methods of management modes. e control group intervened with traditional management. e traditional management mode mainly includes manual registration of the identity of surgical staff, entering and leaving the operating room, receiving surgical clothing, and other medical behaviors. Drugs, equipment, and dressings in the operating room are kept in fixed locations by special personnel and checked regularly. Sterilization items in the operating room were marked with sterilization dates. e number of surgical instruments was counted and recorded by operators, and the operating room nursing staff before and after the operation was to ensure the normal operation. e observation group adopted the intelligent management system. (I) Surgical clothing management automation recorded operators' information to realize intelligent collection and recycling of clothes and shoes in the operating room. e management system tracked the washing and disinfection and used the status and inventory of surgical clothing and shoes in the operating room to remind logistics personnel to add or clean surgical clothing and shoes in time. At the same time, for efficient management level, the whole process of closed-loop management is required for surgical clothing and shoes. (II) Operation staff access control: intelligent identification of operating room medical staff strictly control the operating room irrelevant medical staff entry to effectively control the risk of infection in the operating room. (III) Operation staff behavior management: the combination of an intelligent management system and enterprise scheduling system accurately records the time point of medical staff entering and leaving the operating room to assure the on-time rate of operation. (IV) Operating room system management: according to the operating room management requirements, the historical data of the system reasonably are stored and maintained regularly to ensure the normal operation of all equipment. V) Operating room audit management: safety checked surgical instruments, medicines, blood transfusions, and consumables to form multidimensional knowledge database for data sharing. ## Observation indexes (I) e qualified recovery rate of surgical clothing was observed before and after the implementation of the intelligent management system. e quality rate of clothing recovery � cases of qualified cases of clothing returning after surgery/cases of medical staff × 100%. (II) Observed the on-time operation opening rate before and after the implementation of the intelligent system, with the operation starting at 8:30 as the standard. (III) Discrepancy before and after the implementation of the operating room nursing quality: five assessment team members with more than 10 years of nursing experience were selected, and the hospital operating room nursing quality questionnaires were used to evaluate the operating room nursing quality, including aseptic operation, surgical instruments for qualified, sterile items based nursing management, nursing documents, and operation 5 projects. e percentage system was adopted for each item, and the higher the score, the higher the quality of nursing in the operating room. (IV) Contrasted the satisfaction of medical staff before and after implementation. e satisfaction of medical staff was evaluated by the self-made satisfaction questionnaire of our hospital, which mainly included three aspects: changing clothes 2 Computational Intelligence and Neuroscience flow schedule, operating room environment, and statistical reports. e total score of each aspect was 0 to 100, and the higher the score is, the higher the satisfaction will be. (V) e patient satisfaction questionnaire made by the hospital was adopted. e total score was 100 points, very satisfied: 90 points, satisfied: 75-90 points, general: 60-75 points, and unsatisfied:<60 points. 2.5. Statistical Analysis. SPSS 22.0 software (SPSS Inc., Chicago, USA) was utilized for statistical analysis of the obtained data. e measurement data were displayed as (X ± S), and t-test was utilized for statistical analysis between the two groups. Counting data were displayed as rate (%), and divergence was performed by X 2 test (Chi-square test). P < 0.05 was considered as a statistically notable divergence. # Results ## Divergence between correct rate of distributing surgical Clothing and Qualified Rate of Clothing Recovery before and after Implementation. e correct rate of distributing surgical clothing and the qualified rate of clothing recovery were both higher than before the implemented intelligent management system, with statistical significance (P < 0.05), as shown in. ## A class iii adverse event is one at does not result in any impairment of the patient's body or function despite the occurrence of false facts. e on-time rate of operation starting after the implementation of the intelligent management system was higher than before implementation, and the divergences were statistically notable (P < 0.05). e detection of potential safety hazards after the implementation of the intelligent management system was higher than before implementation, and incidences of Class III adverse events were lower after implementation compared to before implementation, but the divergence was not statistically notable (P > 0.05) as shown in. ## Divergence of surgical items and equipment of medical staff in operating room. e accurate positioning of surgical items and the accurate statistics of equipment use time after the implementation of intelligent management system were higher than before implementation, and the divergences were statistically notable (P < 0.05). e prepared surgical items incorrectly were lower after implementation compared to before implementation (P < 0.05), while the sterile items expire was lower after implementation compared to before implementation but the divergence was not statistically notable (P > 0.05) as shown in [fig_ref] Table 3: Divergence of surgical items and equipment of medical staff in operating room [/fig_ref]. ## Divergence of nursing quality before and after Implementation. e qualified preparation of equipment, qualified preparation of aseptic materials qualified, emergency supplies intact, and qualified nursing technique operation were higher than before implementation, and the divergences were statistically notable (P < 0.05) as shown in [fig_ref] Table 4: Divergence of nursing quality before and after implementation [/fig_ref]. ## Discrepancy of performance appraisal score of operating room hospital. e work motivations of nurses were promoted after implementation compared to before implementation (P < 0.05). e job responsibility and the service attitudes were higher than before implementation; however, the divergences were without statistically notable (P > 0.05) as shown in [fig_ref] Table 5: Discrepancy of performance appraisal score of operating room hospital [/fig_ref]. ## Discrepancy of satisfaction of medical staff before and after implementation. e very satisfied number of nurses was more than before implementation, whereas the result did not reach statistical significance, and the divergences were statistically notable (P > 0.05). In addition, the satisfied number of nurses was more than before implementation, and the unsatisfied number of nurses was less than before implementation as shown in [fig_ref] Table 6: Discrepancy of satisfaction of medical staff before and after implementation [/fig_ref]. ## Discrepancy of data between control group and observation Group. 180 surgical patients were randomly divided into control group and observation group. e control group included 53 males and 37 females, and the observation group included 57 males and 33 females. ere was no statistical divergence in baseline data between the two groups as shown in [fig_ref] Table 7: Discrepancy of data between control group and observation group [/fig_ref]. ## Comparison of satisfaction of patients before and after Implementation. e very satisfied number of patients was more than before implementation, while the result did not reach statistical significance, and the divergences were statistically notable (P > 0.05). In addition, the satisfied number of nurses was more than before implementation, and the unsatisfied number of nurses was less more than before implementation as shown in [fig_ref] Table 8: Comparison of satisfaction of patients before and after implementation [/fig_ref]. # Discussion In recent years, due to the continuous improvement of people's living standards and the continuous development of medical technology, patients have higher requirements for the quality of surgery, especially for the surgical staff and surgical environment [bib_ref] Patient-specific surgical guidance system for intelligent orthopaedics, Kunz [/bib_ref]. It is widely known that the more personnel entering the operating room, the greater the impact on the operating room environment. erefore, the entry and exit of personnel in the operating room must be strictly controlled. e traditional setting of full-time personnel to check and register the medical staff participates in the operation and issues the key to the locker of changing clothes and shoes. With the gradual development of the intelligent operating room system, the introduction of information technology into operating room management has become the trend of operating room construction [bib_ref] Artificial intelligence: a new tool in operating room management. Role of machine..., Bellini [/bib_ref] [bib_ref] Operating room efficiency and hospital capacity: factors affecting operating room use during..., Mcgowan [/bib_ref] [bib_ref] Hybrid simulation: bringing motivation to the art of teamwork training in the..., Kjellin [/bib_ref] [bib_ref] Surgery scheduling optimization considering real life constraints and comprehensive operation cost of..., Xiang [/bib_ref]. e intelligent surgical management system not only effectively controls the entry and exit of operating room Computational Intelligence and Neuroscience [bib_ref] Digital patient models based on Bayesian networks for clinical treatment decision support, Cypko [/bib_ref]. e intelligent management system is of great help to the hospital to improve the regional management of operating room and standardize the behavior of medical staff [bib_ref] Implementation of modern operating room management -experiences made at an university hospital, Hensel [/bib_ref]. e automatic and intelligent management of the distribution and recovery of operating clothes and shoes plays an important role in the management of the operating room [bib_ref] Implementing operating room management science: from the bench to the scheduling office, Macario [/bib_ref]. Traditional operating room personnel receives surgical clothes and shoes through the manual record, while the surgical intelligent clothes sending machine is customized and developed based on the actual needs of the hospital. e dispensing machine is automatically associated with the operation schedule record of the hospital information system to realize the automatic dispensing of surgical clothes according to the size and effectively avoid the wrong dispensing of surgical clothes size. e intelligent management system accelerated the turnover of surgical clothing via optimizing the changing process and simplified the counting process through the closed-loop tracking of washing, disinfection, receiving, and inventory of surgical clothing, which was more conducive to hospital logistics management. What is more, the application of smart locker management also cultivated the habit of utilizing the wardrobe according to regulations and returning clothes in time, and the dressing recovery rate of medical staff was memorably boosted. e application of an intelligent management system not only lessened the workload of cleaning staff but also enhanced the satisfaction of medical staff to the changing process and operating room environment, bringing a better sense of experience to medical staff. e on-time opening rate of operation is one of the indexes to evaluate the medical staff's behavior standard [bib_ref] Surgical workflow simulation for the design and assessment of operating room setups..., Neumann [/bib_ref]. Before the application of the intelligent management system in the operating room, the operation is likely to be delayed due to the weak time concept of the operating room medical staff. In addition, there are often many and miscellaneous departments involved in the operation, and the operating room itinerant nurses often need to contact anesthesiologists and surgeons, which not only increases the working burden of the itinerant nurses but also increases the hidden danger of operation safety [bib_ref] Device-and system-independent personal touchless user interface for operating rooms : one personal..., Ma [/bib_ref]. After the application of the intelligent management system, the intelligent management system plays a good reminder role for the operating room medical staff through the planning of the operating room time and the operation information push function and effectively improves the on-time operation opening rate. Meanwhile, it improves the management level and service level of the hospital operating room and further effectively improves the medical behavior of medical staff and the quality of work [bib_ref] Integrating robotic technology into the operating room, Coon [/bib_ref]. e operation desk information can be sent to the nurse station in the ward, which is beneficial for nurses to prepare related work in advance. e operation desk information can also be pushed by the operation desk information, which ensures that the operation doctors can get the operation desk information in time, so as to arrange the time and work reasonably and avoid the operation delay due to other work. ere are many kinds of equipment and sterile items in the operating room. In order to improve the precision of equipment management and timely statistics on the use, recovery, and storage of sterile items, the intelligent management system can scan these equipment and sterile items to realize information sharing [bib_ref] Environmental controls in operating theatres, Dharan [/bib_ref]. is study shows that the intelligent management system has the function of recording and identifying, which can effectively manage sterile items and surgical equipment, accurately count the use time of equipment and sterile dressing cycle, and effectively reduce the expiration and failure rate of sterile items. At the same time, the intelligent management system has a verification function, which greatly reduces the incidence of medical errors, ensures the smooth development of surgery, and directly ensures the safety of surgery. Intelligent management system to adhere to the quality improvement as the basic concept can improve the medical staff's work initiative and stimulate the sense of responsibility. is study shows that after the application of intelligent management system, the performance evaluation score is significantly improved, and the distribution of performance evaluation can be reasonably completed in a short time, which is conducive to improving the satisfaction of medical staff. In addition, an intelligent operating room can fully integrate patient information, ensure accurate Computational Intelligence and Neuroscience operation, and reduce adverse events caused by improper operation of nursing staff, so as to ensure service quality and improve patient satisfaction. # Conclusion Taken together, the efficiency of medical staff has been improved by intelligent management systems, which have changed the original manual management model. e intelligent management system can effectively improve the ontime operation opening rate, optimize the dressing process, improve the quality of nursing in the operating room, and reduce the occurrence of adverse events in the operating room, which is conducive to enhancing the satisfaction of medical staff and patients. Data Availability e datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. Ethical Approval e authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. e study was carried out in accordance with the ethical guidelines of the Declaration of Helsinki (as revised in 2013). ## Conflicts of interest e authors declare that they have no conflicts of interest. [table] Table 3: Divergence of surgical items and equipment of medical staff in operating room (n, %). [/table] [table] Table 4: Divergence of nursing quality before and after implementation (n, %). [/table] [table] Table 5: Discrepancy of performance appraisal score of operating room hospital (n, %). [/table] [table] Table 6: Discrepancy of satisfaction of medical staff before and after implementation (n, %). [/table] [table] Table 8: Comparison of satisfaction of patients before and after implementation (n, %). [/table] [table] Table 7: Discrepancy of data between control group and observation group (‾X ± S). [/table]

Easy and Versatile Synthesis of Bulk Quantities of Highly Enriched 13C-Graphene Materials for Biological and Safety Applications [bib_ref] Scalable production of large quantities of defect-free few-layer graphene by shear exfoliation..., Paton [/bib_ref] [bib_ref] Highly Decoupled Graphene Multilayers: Turbostraticity at its Best, Mogera [/bib_ref] [fig_ref] Table S2: Table of the different yields of FLG obtained using the different conditions... [/fig_ref] [fig] Figure S2, Figure S3, Figure: TGA of non-graphitized and graphitized 12 C carbon nanofiber. XPS of a) pristine and graphitized 12 C and b) 13 C carbon nanofiber. S4.a) XPS of a) pristine and graphitized 12 C and b) 13 C carbon nanofiber. [/fig] [fig] Figure S5: TEM image of a) 12 C non-graphitized and b) graphitized nanofiber, c) width and d) length size of 12 C carbon nanofibers. [/fig] [fig] Figure S8: TEM Image and size distribution of graphene obtained by the exfoliation of 12 C graphitized carbon nanofibers: a, d) 12 C-GFLG-1, b, e) 12 C-GFLG-2, c, f) 12 C-GGO. [/fig] [fig] Figure S9: a) Raman spectroscopy and b) Thermogravimetric Analysis of 12 C graphene oxide ( 12 C-GGO). [/fig] [table] Table S2: Table of the different yields of FLG obtained using the different conditions of exfoliation. [/table]

Synthesis of Quinolin-2-one Alkaloid Derivatives and Their Inhibitory Activities against HIV-1 Reverse Transcriptase Based on an established common pharmacophore of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNTTIs), a series of quinolin-2-one derivatives were synthesized and assayed for their in vitro activities against HIV-1 reverse transcriptase (RT) for the first time. Some of the tested compounds were active against HIV-1 RT. Compounds 4a2 and 4d2 showed inhibitory activities with IC 50 values of 0.21 and 0.15 μM, respectively, with a mode of interaction with RT residues of the allosteric pocket similar to that of efavirenz. # Introduction AIDS (Aquired Immune Deficiency Syndrome), caused by human immunodeficiency virus (HIV), a RNA dependent retrovirus, remains one of the major causes of death in the World. HIV-1 reverse transcriptase (RT) is one of the enzymes crucial for the HIV virus replication cycle. Upon entering a host cell, the single stranded viral RNA is converted to double-stranded DNA catalyzed by RT and then the virus DNA inserts into the genome of the host cell. Inhibition of reverse transcriptase (RT), the HIV-encoded polymerase which directs both RNA and DNA synthesis, has been proven to be one of the most effective ways to block viral multiplication [bib_ref] Twenty-six years of anti-HIV drug discovery: Where do we stand and where..., Mehellou [/bib_ref] [bib_ref] Design, molecular modeling, synthesis, and anti-HIV-1 activity of new indolyl aryl sulfones...., Ragno [/bib_ref] [bib_ref] Discovery of novel benzimidazolones as potent non-nucleoside reverse transcriptase inhibitors active against..., Barreca [/bib_ref] [bib_ref] Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: Role of two..., Regina [/bib_ref] [bib_ref] Novel indazole non-nucleoside reverse transcriptase inhibitors using molecular hybridization based on crystallographic..., Jones [/bib_ref]. Based on the ready availability of X-ray crystal structures of inhibitor-HIV RT complexes, Freeman and colleagues have identified the common pharmacophore N-(4-chlorophenyl)acetamide (1) from an array of known non-nucleoside reverse transcriptase inhibitors [bib_ref] Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance..., Freeman [/bib_ref] such as efavirenz, DPC961, HBY097 and the newly reported benzoimidazol-2-one [bib_ref] Computational strategies in discovering novel non-nucleoside inhibitors of HIV-1 RT, Barreca [/bib_ref] and dihydroquinoxalin-2-one [bib_ref] Design, synthesis, and structure-activity relationships of 1,3-dihydrobenzimidazol-2-one analogues as anti-HIV agents, Monforte [/bib_ref] NNTRIs [fig_ref] Figure 1: NNRTI pharmacophore N- [/fig_ref]. A series of substituted 2-quinolones (2, [fig_ref] Figure 2: quinolin-2-one NNRTIs and newly designed derivatives [/fig_ref] were synthesized and evaluated as HIV-1 inhibitors in Freeman's research. As to natural products, a similar quinolin-2-one alkaloid 3 isolated from Euodia roxburghiana reported by McCormick also showed inhibitory activity with an IC 50 of 8 μM in an HIV-1 reverse transcriptase (RT) assay [bib_ref] HIV inhibitory natural products. 26. quinoline alkaloids from Euodia roxburghiana, Mccormick [/bib_ref]. Based on the pharmacophore established by Freeman, we designed and synthesized a series of 4-substituted quinolin-2-one alkaloids 4 and 5, as shown in [fig_ref] Figure 2: quinolin-2-one NNRTIs and newly designed derivatives [/fig_ref]. We envisaged that replacement of the cyclopropylethynyloxy moiety with a hydrophobic aromatic ring would retain the inhibitory activity against HIV-1 RT [bib_ref] Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: Role of two..., Regina [/bib_ref] [bib_ref] Synthesis and biological evaluation of C-5 methyl substituted 4-arylthio and 4-aryloxy-3-Iodopyridin-2(1H)-one type..., Guillemont [/bib_ref] [bib_ref] Pyrazole NNRTIs 1: Design and initial optimisation of a novel template, Mowbray [/bib_ref] [bib_ref] Synthesis and biological evaluation of N4-(hetero)arylsulfonylquinoxalinones as HIV-1 reverse transcriptase inhibitors, Xu [/bib_ref] [bib_ref] Structural basis for the improved drug resistance profile of new generation benzophenone..., Ren [/bib_ref]. In compounds 4, the group Y could be oxygen, sulfur, sulfinyl or sulfonyl units, which were flexible linkers to connect the hydrophobic phenyl group with the quinolin-2-one scaffold. To identify the importance of the linker, compounds 5 were synthesized with an aromatic ring directly connected to quinoline-2-one scaffold. In this paper, we report the synthesis of compounds 4 and 5 and the evaluation of the synthesized compounds for their in vitro inhibitory activities against HIV-1 RT for the first time. # Results and discussion ## Chemistry The syntheses of compounds 4 is summarized in Schemes 1 and 2, respectively. 3-Hydroxyquinolin-2-one (8, Scheme 1) has been the subject of intensive synthetic studies for a long time. Among the synthetic strategies used is the Friedel-Crafts reaction [bib_ref] Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance..., Freeman [/bib_ref]. Freeman reported that a treatment with aniline or 4-chloroaniline 6 and excess of diethyl malonate in diphenyl ether at 250 °C for 24 h afforded cyclized compound 8 directly. But in our study, target compound 8 was not obtained under these conditions and only some amide intermediates could be detected by HPLC-ESIMS. We thus irradiated a mixture of aniline and diethylmalonate (2:1 molar ratio) under microwaves and prepared corresponding dimalonamide 7 in good yield. Heating N,N'-di(4-chlorophenyl)malonamide in polyphosphoric acid (PPA) at 140-150 °C afforded the 4-hydroxy-2-quinolone 8 (Scheme 1) [bib_ref] Synthesis of 6-chloro and 6-fluoro-4-hydroxyl-2-quinolone and their azo disperse dyes, O&apos;llah [/bib_ref]. Refluxing compound 8 in POCl 3 provided 2,4,6-trichloroquinoline 9 in good yield. A phenylthio group was then installed on the quinoline ring to afforded compounds 10, which were converted to target compounds 4a through a microwave assisted hydrolysis in mixture of TFA and HCl. Compounds 4a were oxidized to target compounds 4b as racemates, and these were then converted to compounds 4c by reaction with another 1.1 equiv. of 3-chlorobenzoperoxoic acid (m-CPBA). The synthesis of compounds 4d shared the same synthetic precursor 2,4,6-trichloroquinoline 9 (Scheme 2). Through treatment of compound 9a with various phenols followed by hydrolysis in acid mixtures, compounds 4d were synthesized in moderate yields using the conditions described above without need for further optimization. The synthetic route of target compounds 5 was summarized in Scheme 3. The synthesis of 5 by various methods has been reviewed. The majority of compounds 13 were prepared by the reaction of aryl esters with ortho-lithiated Boc-protected 4-chloroaniline via the formation of the dianion species with t-BuLi (2.2 equiv) followed by deprotection of the Boc group (Scheme 3) [bib_ref] Beyond thermodynamic acidity: A perspective on the complex-induced proximity effect (CIPE) in..., Whisler [/bib_ref] [bib_ref] The synthesis and structure-activity relationships of 4-aryl-3-aminoquinolin-2-ones: A new class of calcium-dependent,..., Hewawasam [/bib_ref]. A tandem amidation/Knoevengel condensation of compounds 13 with ethyl acetate gave compounds 5 in good yields. ## Enzymatic activities Compounds 4a-4d were tested in RT inhibition assays and proved to be active as inhibitors of HIV-1 RT [fig_ref] Table 1: HIV-1 RT inhibitory activities of compounds 4-5 [/fig_ref]. The enzymatic data highlighted that derivative 4a2 (IC 50 = 0.21 μM) and 4d2 (IC 50 = 0.15 μM) turned out to be more active than 4a3 (IC 50 = 13 μM), respectively, and suggested that the presence of a chlorine substituent at C-6 may lead to an increase of potency and influence the affinity to the enzyme. This finding is in agreement with the N-(4-chlorophenyl)acetamide pharmacophore established by Freeman, and new type of anti-HIV-1 RT compounds could be designed based on the pharmacophore model. Cl [formula] O H 3.0 4d2 Cl O 3,5-CH 3 0.15 5a Cl / H >100 5b Cl / 2-F >100 5c [/formula] Cl / 2-Cl >100 a All values are the mean of two independent experiments; b The inhibition of recombinant HIV-1 RT activity was performed with a commercially available ELISA kit (Roche) according to the instructions of the manufacturer. IC 50 = effective concentration that inhibits 50% of HIV-1 RT; c Efavirenz: an HIV-1 RT inhibitor used as positive control. IC 50 = 6 nM. In addition, the type of linker which connected the aromatic ring with the quinoline scaffold might be crucial for potent anti-HIV-1 RT activity in the title compounds. Comparing compounds 4a2 (IC 50 = 0.21 μM), 4b2 (IC 50 = 2.6 μM) and 4c2 (IC 50 = 10 μM), a decrease of activity against RT could be observed as the degree of oxidation of the sulfur atom increased. The role of the substituents of the phenyl ring on the biological activities was also investigated, as shown in [fig_ref] Table 1: HIV-1 RT inhibitory activities of compounds 4-5 [/fig_ref]. In compounds 4a1 (IC 50 = 5.6 μM) and 4a2 (IC 50 = 0.21 μM), two hydrophobic methyl group on the phenyl ring led to potent enzyme activity, and may contribute to the ability to bind with the hydrophobic site of RT. In contrast, compounds 5a-5c exhibited obviously decreased activities towards the enzyme. We envisagd that hydrophobic interaction ability was influenced by the rigid conjugation system between the phenyl ring and the quinolin-2-one scaffold. ## Docking studies To investigate the binding mode of the synthesized quinolin-2-ones, computational modeling was performed using CDOCK. The CHARMM force field was used for the energy minimizations in the docking process. The coordinates of the RT-wfavirenz complex (PDB code: 1FK9) [bib_ref] Synthesis and biological evaluation of N4-(hetero)arylsulfonylquinoxalinones as HIV-1 reverse transcriptase inhibitors, Xu [/bib_ref] [bib_ref] Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations..., Ren [/bib_ref] were downloaded, and the molecular structure of the most potent compounds 4a2 and 4d2 were docked into its active site. The best docked conformation of compounds 4a2 and 4d2 were shown in [fig_ref] Scheme 3: Synthesis of target compounds 5 [/fig_ref] , together with the experimental position of efavirenz and the 3D common-feature pharmacophore model. Compounds 4a2 and 4d2 interact with RT residues of the allosteric pocket in a fashion similar to known NNRTIs. The p-chloroaniline moiety has a hydrophobic contact with the surrounding residues such as Tyr318, Pro236, Leu234 and Val106. The carboxamide group of the molecule interacts with the main-chain of Lys101 by hydrogen bond interactions. The 3,5-dimethylphenyl fragment occupies a hydrophobic pocket consisting of Pro95, Tyr181, Tyr188 and Trp229, which is occupied by the cyclopropyl-propynyl of efavirenz in the complex. One of the two methyl groups on the benzene ring adopts a similar orientation to the cyclopropyl group of efavirenz. ## Experimental ## Chemistry # Instruments and materials Column chromatography silica gel (200-300 mesh) and TLC plate (Qingdao Meijin Chemical Inc.; Qingdao; China); MS data were obtained on an AutoSpec-3000 or Agilent HPLC-ESIMS; 1 H-NMR spectra were recorded on Bruker 300M or DRX-500 spectrometers and chemical shifts were given in δ with TMS as an internal reference. All the reagents are commercial available. Tetrahydrofuran (THF) was refluxed over Na before use. ## General procedure for preparation of compounds 4 ## Synthesis of intermediates 7 Aniline or 4-chloroaniline (100 mmol), and diethyl malonate (50 mmol) were properly mixed in a 250 mL beaker. The obtained mixture was irradiated in a microwave oven at the power of 320 W for 5 min. After irradiation, the crude product was recrystallised in ethanol to afford the desired product. ## Synthesis of intermediates 9 A mixture of 8 (84.47 mmol) in POCl 3 (150 mL) was heated under reflux for 3h. After cooling down, the mixture was slowly added to crushed ice while shaking. The precipitate was collected by filtration and washed several times with water. The product was dried under vacuum overnight to give the products as white solid which are pure enough for further use. ## Synthesis of intermediates 10 To a solution of 9a or 9b (10.0 mmol) in DMF (10 mL) was added Et 3 N (2.78 mL, 20 mmol) and a solution of thiol (10 mmol) in DMF (10 mL) dropwise at 0 °C. The reaction was warmed up after 10 min and stirred overnight at rt. 200 mL of EtOAc was added to the mixture and washed with water and brine successively. The organic layer was separated and dried over Na 2 SO 4 . The product was purified on a column after removal of the solvent under vacuum. In most of cases, less than 7% of dithio-substituted quinoline was formed. ## Synthesis of target compounds 4a A solution of compound 10 (0.50 mmol) in TFA (2 mL) and HCl (6 N, 2 mL) was heated under microwave radiation at 120 °C for 20 min. After cooling down, water (20 mL) was added. The precipitate was collected by filtration and washed with water several times. The product was air dried (71-85%) and was pure enough without further purification. The solubility of the products in organic solvents is poor. ## 6-chloro-4-(phenylthio)quinolin-2(1h)-one ## Synthesis of target compounds 4b To a solution of compound 4a (0.25 mmol) in a mixture of CH 2 Cl 2 and MeOH (50 mL) was added 1.1 equiv. of 3-chlorobenzoperoxoic acid. After stirring overnight, water (50 mL) was added. The mixture was extracted with CH 2 Cl 2 and evaporated to give a residue which were further purified on silica gel column to give compounds 4b (66-75%). ## 6-chloro-4-(phenylsulfinyl)quinolin-2(1h)-one ## Synthesis of target compounds 4c To a solution of compound 4b (0.2 mmol) in a mixture of CH 2 Cl 2 and MeOH (50 mL) was added 1.1 equiv. 3-chlorobenzoperoxoic acid. After stirring overnight, 50 mL of water was added. The mixture was extracted with CH 2 Cl 2 and evaporated to give a residue which were further purified on silica gel column to give compound 4b (60-66%). ## 6-chloro-4-(phenylsulfonyl)quinolin-2(1h)-one ## Synthesis of target compounds 4d To a solution of 9a (10.0 mmol) in DMF (10 mL) was added Et 3 N (2.78 mL, 20 mmol) and a solution of phenol (10 mmol) in DMF (10 mL) dropwise at 0 °C. The reaction was warmed up after 10 min and stirred for 24h at rt. 200 mL of EtOAc was added to the mixture and washed with water and brine successively. The organic layer was separated and dried over Na 2 SO 4 . The product 11 was purified on column after removal of solvent under vacuum with yield of 75-86%. A solution of compound 11 (0.50 mmol) in TFA (2 mL) and HCl (6 N, 2 mL) was then heated under microwave radiation at 120 °C for 20 min. After cooling down, 20 mL of water was added. The precipitate was collected by filtration and washed with water for several times. The product was air dried (71-85%) and was pure enough without further purification. [fig_ref] Figure 1: NNRTI pharmacophore N- [/fig_ref] , DIEA (0.12 mol) and toluene (40 mL) were placed in a 100 mL flask and stirred at rt, then Boc 2 O (26 g, 0.12 mol) was added, and the solution was stirred at rt until the completion of the reaction monitored by TCL. Then the solution was poured into water and extracted with DCM and then the organic layer was washed by water and dried over anhydrous Na 2 SO 4 . Then the organic solvent was removed under vacuum to give crude product which was further purified on Si gel column chromatography and eluted with petroleum ether (PET)-EtOAc (6:1) to give the compound tert-butyl 4-chlorophenylcarbamate (12) The combined organic layer was washed with brine and dried over anhydrous Na 2 SO 4 and then evaporated to give crude N-Boc protected 13. Without further purification, the crude product was dissolved in 30 mL EtOH, and then 3 mol/L HCl solution (10 mL) was added. The mixture was refluxed until the completion of the reaction. Then the solution was adjusted to pH 8 using saturated NaHCO 3 solution and extracted with DCM successively. The organic layer was washed with brine and dried over anhydrous Na 2 SO 4 and then evaporated to give a residue which was further purified on Si CC to give compound 13 (3.46 g, yield: 30%). ## Synthesis of compounds 5 Compound 5a was taken as an example. A 30 mL, 2-necked flask was charged with compound 13a (1 mmol, 231 mg) and THF (5 mL). The resulting solution was cooled to 0 °C and LiHMDS (6 mL, 1M in THF) was added over 5 min. The internal temperature was controlled <5 °C for 10 min, then EtOAc (144 mg, 2 mmol) was added over 1 min. The reaction solution was allowed to warm up to rt and stirred at rt for 2h. Then water (5 mL) was added, and the reaction mixture was stirred at rt for another 24 h. The solution was poured into 50 mL ice cooled water, and a water suspension formed. The suspension was filtered and the solid was further purified on Si CC eluted with CHCl 3 /CH 3 OH (97:3) to give white amorphous powder 5a (212 mg, yield: 83%). ## General procedure for hiv-1 rt inhibitory assay HIV-1 reverse transcriptase (RT) activity was measured using a commercially available ELISA RT kit (Roche) according to the instructions of the manufacturer. The compounds were incubated with DIG-labeled reaction mixture at 37 °C for 2 h, then anti-DIG-POD solution was added, followed by substrate ABTS. Efaviren was used as a positive control. The absorbency at 405 nm/490 nm (A405/490) was read on Bio-Tek ELx 800 ELISA reader. ## General procedures for docking study CDOCK was used for the docking study. The CHARMM force field was used for the energy minimizations in the docking process. The X-ray crystal structure of reverse transcriptase complexed with efavirenz was retrieved from PDB (PDB code: 1FK9). The active site was defined as all residues within 6.5 Å radius of the cocrystallized efavirenz. Starting from the ligand configuration, a set of 10 different orientations was randomly generated and ranked according to their binding free energy. The docking mode was chosen on the basis of binding affinity rank. # Conclusions In summary, a group of quinoneline-2-one analogues with activity against HIV-1 RT were synthesized based on the SAR established by Freeman and known crystal structures. The SAR and docking studies of the titled compounds were analyzed. Analogues 4a2 and 4d2 demonstrated the most activity among these analogues with a similar mode of interaction with RT residues of the allosteric pocket as known NNRTIs. Further structure modifications and cell-based in vitro anti-HIV-1 assay studies of these quinolin-2-one derivatives will be reported in due course. [fig] Figure 1: NNRTI pharmacophore N-(4-chlorophenyl)acetamide (1) and related anti-HIV-1 RT active compounds. [/fig] [fig] Figure 2: quinolin-2-one NNRTIs and newly designed derivatives. [/fig] [fig] Scheme 1: Synthesis of target compounds 4a-4c. Reagents and conditions: (a) diethylmalonate, MW (320 W), 10 min; (b) PPA, 140-150 °C, 6 h; (c) POCl 3 , reflux, 3 h; (d) benzenethiol, 1.0 eq, Et 3 N, DMF, 0 °C→rt, overnight; (e) TFA, HCl, MW (320 W), 20 min; (f) m-CPBA, 1.1equiv., CH 2 Cl 2 , MeOH, rt; (g) m-CPBA, 1.1equiv., CH 2 Cl 2 , MeOH, rt. [/fig] [fig] Scheme 2: Synthesis of target compounds 4d. Reagents and conditions: (h) phenol, 1.0 eq. Et 3 N, DMF, 0 °C→rt, overnight; (i) TFA, HCl, MW (320 W), 20 min. [/fig] [fig] Scheme 3: Synthesis of target compounds 5. [/fig] [fig] Figure 3: CDOCK-modeled binding mode of 4a2 (left) and 4d2 (right) in comparison with the crystal structure (1FK9 in PDB) of efavirenz (orange colored carbon atoms). The key hydrogen bond is illustrated with green lines. [/fig] [fig] bis 4 -: Chlorophenyl)malonamide (7a): yield 95%, white solid, ESIMS: m/z: 323 (M+H) + Diphenylmalonamide (7b): yield 91%, white solid, ESIMS: m/z: 255 (M+H) + 3.1.2.2. Synthesis of Intermediates 8A mixture of 7a or 7b (2.0 mmol) with 5-6 times by weight polyphosphoric acid were stirred in an oil bath at 140-150 °C for 6 h. Then the mixture was cooled, diluted with water and the resultant gum solidified by standing over night. The solid was filtered and dried in air. It was recrystallised from ethanol to afford corresponding hydroxyquinolones as creamy crystals.6-Chloro-4-hydroxyquinolin-2(1H)-one (8a): white solid, yield: 82%, 1 H-NMR (300 MHz, DMSO-d 6 , δ ppm): 5.80 (s, 1H), 7.68 (s, 1H), 7.27 (dd, 1H, J = 8.6, 4.8 Hz), 7.53 (d, 1H, J = 8.6 Hz), 11.35 (brs, 1H, NH). 4-Hydroxyquinolin-2(1H)-one (8b): white solid, yield: 85%, 1 H-NMR (300 MHz, DMSO-d 6 , δ ppm): 5.83 (s, 1H), 7.51-7.68 (m, 3H), 7.23 (d, 1H, J = 8.0 Hz), 11.33 (brs, 1H, NH). [/fig] [fig] 2, 4 , 6 -: Trichloroquinoline (9a): white solid, yield 70%, 1 H-NMR (500 MHz, CDCl 3 , δ ppm): 8.00 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H); ESIMS: m/z: 232 (M+H) + . 2,4-Dichloroquinoline (9b): white solid, yield 66%, 1 H-NMR (500 MHz, CDCl 3 , δ ppm): 8.16 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.47 (s, 1H); ESIMS: m/z: 198 (M+H) + . [/fig] [fig] 2, 6 -: Dichloro-4-(phenylthio)quinoline (10a): amorphous powder, yield: 69%. 1 H-NMR (300 MHz, CDCl 3 , δ ppm): 8.11 (s, 1H), 7.88-7.50 (m, 2H), 7.18-7.09 (m, 5H), 6.72 (s, 1H); ESIMS: m/z: chloro-4-(3,5-dimethylphenylthio)quinoline (10c): amorphous powder, yield: 59%. 1 H-NMR (300 MHz, CDCl 3 , δ ppm): 8.23 (d, J = 8.0 Hz, 1H), 7.99-7.57 (m, 3H), 7.23 (s, 1H), 7.21 (s, 1H), 7.16 (s, 1H), 6.63 (s, IH), 2.33 (s, 6H); ESIMS: m/z: 282 (M+H) + . [/fig] [fig] 6 -: Chloro-4-phenoxyquinolin-2(1H)-one (4d1): amorphous powder, yield: 71%. 1 H-NMR (300 MHz, DMSO-d 6 , δ ppm): 11.94 (bs, 1H), 7.71 (s, 1H), 7.60-7.10 (m, 2H), 7.01-6.67 (m, 5H), 5.70 (s, 1H); ESIMS: m/z: 272 (M+H) + ; HRESIMS: calc for C 15 H 11 ClNO 2 [M+H] + 272.0478, found 272.0470. 6-Chloro-4-(3,5-dimethylphenoxy)quinolin-2(1H)-one (4d2): amorphous powder, yield: 77%. 1 H-NMR (300 MHz, DMSO-d 6 , δ ppm ): 11.64 (bs, 1H)7.70 (s, 1H), 7.65-7.31 (m, 2H), 6.89 (s, 2H), 6.93 (s, 1H), 5.72 (s, 1H), 2.33 (s, 6H); ESIMS: m/z: 300 (M+H) + ; HRESIMS: calc for C 17 H 15 ClNO 2 [M+H] + 300.0791, found 300.0775. [/fig] [fig] 2 -: Amino-5-chlorophenyl)(phenyl)methanone (13a): 1 H-NMR (DMSO-d 6 , δ ppm): 7.64-7.33 (m, 7H), 6.99 (d, J = 8.8 Hz, 1H), 6.08 (brs, 2H, NH 2 ); positive FABMS m/z (%): 232 [M+1] + (100). [/fig] [table] Table 1: HIV-1 RT inhibitory activities of compounds 4-5. a [/table]

An Update on the Implications of New Psychoactive Substances in Public Health Citation: Simão, A.Y.; Antunes, M.; Cabral, E.; Oliveira, P.; Rosendo, L.M.; Brinca, A.T.; Alves, E.; Marques, H.; Rosado, T.; Passarinha, L.A.; et al. An # Brief introduction According to the United Nations Office on Drugs and Crime (UNODC), new psychoactive substances (NPS) are classified as "substances of abuse, either in a pure form or a preparation, that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat". Essentially the term "new" is used to report that a certain substance has emerged on the market illegally and is being used recreationally rather than medically. Moreover, these substances can also be called "designer drugs", since they are designed to mimic the effects of classical illicit substances that follow strict regulations from governmental entities and that are scheduled under drug control conventions [bib_ref] New Psychoactive Substances: Evolution in the Exchange of Information and Innovative Legal..., Varì [/bib_ref]. In fact, the global occurrence of NPS poses major concerns and challenges for law enforcement agencies, but also for public health, due to constant diversification and growth. The more chemical and structural modifications are performed in a molecule, the more difficult it is for drug control legislations to track down these substances. To aggravate matters, NPS are often produced The two main structural groups that form the piperazine-based compounds are 1benzylpiperazine and 1-phenylpiperazine [bib_ref] Designer drugs: Mechanism of action and adverse effects, Luethi [/bib_ref] [bib_ref] Piperazine compounds as drugs of abuse, Arbo [/bib_ref] [bib_ref] Piperazine designer drugs elicit toxicity in the alternative in vivo model Caenorhabditis..., Souto [/bib_ref] [bib_ref] Piperazine derivatives as dangerous abused compounds, Welz [/bib_ref] [bib_ref] Current awareness of piperazines: Pharmacology and toxicology, Elliott [/bib_ref] [bib_ref] Stability of synthetic piperazines in human whole blood, Lau [/bib_ref] [bib_ref] Analytical toxicology of emerging drugs of abuse-An update, Meyer [/bib_ref] [bib_ref] Chemical classification of new psychoactive substances (NPS), Zapata [/bib_ref] [bib_ref] Rapid determination of piperazine-type stimulants in human urine by microextraction in packed..., Moreno [/bib_ref]. In the second half of the 1990s, piperazine designer drugs emerged in the illicit market, but the United States and Scandinavia were the only regions reporting high consumption rates [bib_ref] Piperazine designer drugs elicit toxicity in the alternative in vivo model Caenorhabditis..., Souto [/bib_ref] [bib_ref] Current awareness of piperazines: Pharmacology and toxicology, Elliott [/bib_ref] [bib_ref] Determination of piperazine derivatives in "legal highs, Byrska [/bib_ref]. N-benzylpiperazine (BZP) was the first consumed derivative of piperazines in 1996 in the United States. It is usually combined in pills with 1-(3-trifluoromethylphenyl)piperazine (TFMPP), giving rise to the much sought-after 3,4methylenedioxymethamphetamine (MDMA) in humans, despite being weaker in comparison [bib_ref] Piperazine compounds as drugs of abuse, Arbo [/bib_ref] [bib_ref] Current awareness of piperazines: Pharmacology and toxicology, Elliott [/bib_ref] [bib_ref] Stability of synthetic piperazines in human whole blood, Lau [/bib_ref]. The appearance of new piperazines in Europe has been irregular, and the first case of piperazine abuse reported in Europe occurred in Sweden in 1999. These were not reported until 2006 by the EU Early Warning System (EWS). Subsequently to their first identification, piperazines appeared and were detected from 2005 to 2019. Only in 2004 they were considered NPS, and the EU proceeded to control their consumption in 2008. In England and Wales, between 2008 and 2009, deaths caused by drugs of abuse mainly involved piperazines. Currently, these compounds have spread worldwide, and around ## Aminoindanes Aminoindanes have been investigated as research chemicals. They can act in the dilation of blood vessels, enabling strong potential for aiding in respiratory processes and thus leading to acting as strong bronchodilators. The first development regarding their research relied on these properties [bib_ref] The metabolic fate of the two new psychoactive substances 2-aminoindane and N-methyl-2-aminoindane..., Manier [/bib_ref]. In 1944, an animal study conducted by Levin et al. demonstrated that 2-AI and other derivatives were more effective vasodilators than L-ephedrine, but without showing the toxicity of other amphetamines [bib_ref] Physiologically active indanamines, Levin [/bib_ref]. In 1961 and 1963, two different research papers investigated the analgesic potential of these compounds [bib_ref] Pharmacology of 2-amino-indane hydrochloride (Su-8629): A potent non-narcotic analgesic, Witkin [/bib_ref] [bib_ref] 2-Aminoindans of pharmacological interest, Solomons [/bib_ref]. In 1973, aminoindanes were also investigated in the context of neurodegenerative diseases. Martin et al. conducted an animal study in which a few aminoindanes derivatives were tested for anti-Parkinson effects, but no evidence that they could reduce or even act on the disease's symptoms was observed in spite of monoamine oxidase (MAO) being inhibited by some compounds [bib_ref] Potential anti-Parkinson drugs designed by receptor mapping, Martin [/bib_ref]. The MAO-B inhibition allows for a longer action of dopamine in the brain, ameliorating Parkinson's symptoms. concluded that the biological effect of aminoindanes is directly related to the position and type of the amine substitute [bib_ref] Discriminant analysis of the relation between physical properties and the inhibition of..., Martin [/bib_ref]. The inhibitory effect of aminoindanes on MAO-B was further investigated by to the identification of a strong inhibitor that did not reveal any cardiovascular or central nervous system (CNS) adverse effects. This compound was rasagiline [fig_ref] Figure 2: Chemical structure of rasagiline [/fig_ref] , which was used in the treatment of Parkinson's disease [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref]. vestigated the analgesic potential of these compounds [bib_ref] Pharmacology of 2-amino-indane hydrochloride (Su-8629): A potent non-narcotic analgesic, Witkin [/bib_ref] [bib_ref] 2-Aminoindans of pharmacological interest, Solomons [/bib_ref]. In 1973, aminoindanes w also investigated in the context of neurodegenerative diseases. animal study in which a few aminoindanes derivatives were tested for anti-Parkinso fects, but no evidence that they could reduce or even act on the disease's symptoms observed in spite of monoamine oxidase (MAO) being inhibited by some compounds The MAO-B inhibition allows for a longer action of dopamine in the brain, ameliora Parkinson's symptoms. concluded that the biological effect of noindanes is directly related to the position and type of the amine substitute [bib_ref] Discriminant analysis of the relation between physical properties and the inhibition of..., Martin [/bib_ref]. The inhibitory effect of aminoindanes on MAO-B was further investigated by K et al. in 1981, leading to the identification of a strong inhibitor that did not reveal cardiovascular or central nervous system (CNS) adverse effects. This compound rasagiline [fig_ref] Figure 2: Chemical structure of rasagiline [/fig_ref] , which was used in the treatment of Parkinson's disease [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref]. Selegiline is an analog of rasagiline, its main metabolites are L-amphetamine an methamphetamine, whereas rasagiline does not metabolize into these compounds [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref] The chemical structure of aminoindanes is very similar to that of amphetamines ure 3). In 1986, Nichols et al. defined a group of psychoactive substances for therapeutic featuring molecules such as MDMA, 3,4-Methylenedioxyamphetamine (MDA), and methylenedioxy-2-aminoindane (MDAI) [bib_ref] Structure-activity-relationships of MDMA and related-compounds-A new class of psychoactivedrugs, Nichols [/bib_ref] [bib_ref] Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butanamine: Representatives of a novel therapeutic class, Nichols [/bib_ref]. The authors conducted a study usin animal model to understand the differences between the use of MDMA and noindanes, such as MDAI. The results pointed out that aminoindanes mimic the effec Selegiline is an analog of rasagiline, its main metabolites are L-amphetamine and Lmethamphetamine, whereas rasagiline does not metabolize into these compounds [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref] [bib_ref] Selegiline and rasagiline: Twins or distant cousins?, Knudsen Gerber [/bib_ref]. The chemical structure of aminoindanes is very similar to that of amphetamines [fig_ref] Figure 3: Chemical structure of amphetamine [/fig_ref]. also investigated in the context of neurodegenerative diseases. animal study in which a few aminoindanes derivatives were tested for anti-Parkinson fects, but no evidence that they could reduce or even act on the disease's symptoms observed in spite of monoamine oxidase (MAO) being inhibited by some compounds The MAO-B inhibition allows for a longer action of dopamine in the brain, ameliora Parkinson's symptoms. concluded that the biological effect of a noindanes is directly related to the position and type of the amine substitute [bib_ref] Discriminant analysis of the relation between physical properties and the inhibition of..., Martin [/bib_ref]. The inhibitory effect of aminoindanes on MAO-B was further investigated by K et al. in 1981, leading to the identification of a strong inhibitor that did not reveal cardiovascular or central nervous system (CNS) adverse effects. This compound rasagiline [fig_ref] Figure 2: Chemical structure of rasagiline [/fig_ref] , which was used in the treatment of Parkinson's disease [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref]. Selegiline is an analog of rasagiline, its main metabolites are L-amphetamine an methamphetamine, whereas rasagiline does not metabolize into these compounds [bib_ref] N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B, Youdim [/bib_ref] The chemical structure of aminoindanes is very similar to that of amphetamines ( ure 3). In 1986, Nichols et al. defined a group of psychoactive substances for therapeutic featuring molecules such as MDMA, 3,4-Methylenedioxyamphetamine (MDA), and methylenedioxy-2-aminoindane (MDAI) [bib_ref] Structure-activity-relationships of MDMA and related-compounds-A new class of psychoactivedrugs, Nichols [/bib_ref] [bib_ref] Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butanamine: Representatives of a novel therapeutic class, Nichols [/bib_ref]. The authors conducted a study usin animal model to understand the differences between the use of MDMA and a noindanes, such as MDAI. The results pointed out that aminoindanes mimic the effec In 1986, Nichols et al. defined a group of psychoactive substances for therapeutic use featuring molecules such as MDMA, 3,4-Methylenedioxyamphetamine (MDA), and 5,6methylenedioxy-2-aminoindane (MDAI) [bib_ref] Structure-activity-relationships of MDMA and related-compounds-A new class of psychoactivedrugs, Nichols [/bib_ref] [bib_ref] Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butanamine: Representatives of a novel therapeutic class, Nichols [/bib_ref]. The authors conducted a study using an animal model to understand the differences between the use of MDMA and aminoindanes, such as MDAI. The results pointed out that aminoindanes mimic the effects of MDMA and other derivatives without neurotoxicity in 5-HT receptors [bib_ref] +)-N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine as a discriminative stimulus in studies of 3,4-methylenedioxy-methamphetamine-like behavioral activity, Oberlender [/bib_ref]. In 2014, Simmler et al. [bib_ref] Pharmacological profiles of aminoindanes, piperazines, and pipradrol derivatives, Simmler [/bib_ref] performed a monoamine transporters (MATs) inhibition test on human embryonic kidney cells and concluded that MDAI, 2-AI, and 5-iodo-2-aminoindane (5-IAI) had lower inhibitory effects than MDMA. Around 2010, and due to their similarity to amphetamines, aminoindanes emerged in the United Kingdom's illicit drug market as a new class of NPS-synthetic aminoindanes posing as alternatives to legislated compounds from other classes. The ability of these compounds to mimic the effects of serotonin-releasing drugs, such as MDMA and other amphetamines, makes them effective alternatives to these drugs. Furthermore, these compounds stimulate the CNS, mediating the action of serotonin, norepinephrine, and dopamine. MDMA arose recent interest for the treatment of post-traumatic stress disorder [bib_ref] The emerging role of psilocybin and MDMA in the treatment of mental..., Gill [/bib_ref]. However, its application in other comorbid psychiatric conditions has been poorly investigated. Some studies have proven pro-cognitive effects, such as improving social connectedness and mood, increased responsiveness, and emotional sensitivity. However, other studies have also proven anti-cognitive effects [bib_ref] The emerging role of psilocybin and MDMA in the treatment of mental..., Gill [/bib_ref]. These synthetic aminoindanes derive from 2-AI [fig_ref] Figure 4: Chemical structures of synthetic aminoindanes [/fig_ref] , some of the most common originating from structural modifications on the 2-AI precursor molecule. Some examples of these molecules are MDAI, 5,6-Methylenedioxy-N-methyl-2-aminoindane (MDMAI), 5-methoxy-6-methyl-2,3-dihydro-1H-inden-2-amine (MMAI), N-methyl-2,3dihydro-1H-inden-2-amine (NM-2-AI), and 5-IAI. such as improving social connectedness and mood, increased responsiveness, and emotional sensitivity. However, other studies have also proven anti-cognitive effects [bib_ref] The emerging role of psilocybin and MDMA in the treatment of mental..., Gill [/bib_ref]. These synthetic aminoindanes derive from 2-AI [fig_ref] Figure 4: Chemical structures of synthetic aminoindanes [/fig_ref] , some of the most common originating from structural modifications on the 2-AI precursor molecule. Some examples of these molecules are MDAI, 5,6-Methylenedioxy-N-methyl-2-aminoindane (MDMAI), 5-methoxy-6-methyl-2,3-dihydro-1H-inden-2-amine (MMAI), N-methyl-2,3dihydro-1H-inden-2-amine (NM-2-AI), and 5-IAI. There is still a lack of general information about these substances, mainly concerning their pharmacological effects, which makes the legislation of these compounds challenging. The UNODC reports that none of these NPS is under international control. In fact, until 2012, aminoindanes were the second smaller NPS group reported to the UNODC, with just 34 reported cases at a global scale against 684 of synthetic cathinones. Synthetic aminoindanes are usually found as powders and crystals, and are consumed through ingestion, snorting, or rectal application. No reported cases of the injection or smoking of these substances exist [bib_ref] ) toxicity: A brief overview and update, Corkery [/bib_ref] [bib_ref] Is the 5-iodo-2-aminoindan (5-IAI) the new MDMA?, Coppola [/bib_ref] [bib_ref] 5-Iodo-2-aminoindan (5-IAI): Chemistry, pharmacology, and toxicology of a research chemical producing MDMA-like..., Coppola [/bib_ref]. Common street names are "MDAI gold" or "Pink Champagnes", the former for MDAI-based substances and the latter for preparations containing 2-AI. Information on the effects and consequences of the consumption of these compounds has been found in forums of users' experiences, such as Erowid. Some known effects are stimulation, empathy, and euphoria, while adverse effects include anxiety, depression, and tachycardia. Users have reported the onset of effects for these substances being about 30 min after oral consumption, and the window for maximum effect is between 45 min and 3 h after ingestion. Manier et al. [bib_ref] The metabolic fate of the two new psychoactive substances 2-aminoindane and N-methyl-2-aminoindane..., Manier [/bib_ref] investigated in vivo and in vitro metabolic pathways for 2-AI and NM-2-AI. The former is metabolized via N-acetylation, while the latter suffers There is still a lack of general information about these substances, mainly concerning their pharmacological effects, which makes the legislation of these compounds challenging. The UNODC reports that none of these NPS is under international control. In fact, until 2012, aminoindanes were the second smaller NPS group reported to the UNODC, with just 34 reported cases at a global scale against 684 of synthetic cathinones. Synthetic aminoindanes are usually found as powders and crystals, and are consumed through ingestion, snorting, or rectal application. No reported cases of the injection or smoking of these substances exist [bib_ref] ) toxicity: A brief overview and update, Corkery [/bib_ref] [bib_ref] Is the 5-iodo-2-aminoindan (5-IAI) the new MDMA?, Coppola [/bib_ref] [bib_ref] 5-Iodo-2-aminoindan (5-IAI): Chemistry, pharmacology, and toxicology of a research chemical producing MDMA-like..., Coppola [/bib_ref]. Common street names are "MDAI gold" or "Pink Champagnes", the former for MDAI-based substances and the latter for preparations containing 2-AI. Information on the effects and consequences of the consumption of these compounds has been found in forums of users' experiences, such as Erowid. Some known effects are stimulation, empathy, and euphoria, while adverse effects include anxiety, depression, and tachycardia. Users have reported the onset of effects for these substances being about 30 min after oral consumption, and the window for maximum effect is between 45 min and 3 h after ingestion. Manier et al. [bib_ref] The metabolic fate of the two new psychoactive substances 2-aminoindane and N-methyl-2-aminoindane..., Manier [/bib_ref] investigated in vivo and in vitro metabolic pathways for 2-AI and NM-2-AI. The former is metabolized via N-acetylation, while the latter suffers hydroxylation. The authors identified the formation of hydroxylamine in the metabolic pathway of this last compound, which can be toxic for humans or animals at high concentrations [bib_ref] Biologic Activity of Hydroxylamine: A Review, Gross [/bib_ref]. Palenicek et al. in 2016 [bib_ref] Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats, Páleníček [/bib_ref] demonstrated that MDAI (administered to rats at 10 mg/kg) was rapidly absorbed in the brain, even though the larger concentration was found in the lungs. The authors observed increased and selective neurotoxicity for this compound. Some studies on animal models stated that the intake of aminoindanes at larger doses increases neurotoxicity and body temperature, which can originate toxic conditions (for instance, serotoninergic syndrome). Aminoindanes act primarily on 5-HT-receptors, and therefore stimulation is not as high as occurs when other compounds (cocaine, MDMA) are used. Thus, users consume higher doses of the compounds or mix them with other stimulant drugs, which has a potential risk of cardio-and neurotoxic conditions [bib_ref] ) toxicity: A brief overview and update, Corkery [/bib_ref] [bib_ref] Synthesis and pharmacological examination of benzofuran, indan, and tetralin analogs of 3,4-(methylenedioxy)amphetamine, Monte [/bib_ref]. in 2012 that MDAI has been linked to several toxic conditions, such as renal and hepatic failure, valvular heart disease, and respiratory distress syndrome [bib_ref] 6-methylenedioxy-2-aminoindane: From laboratory curiosity to 'legal high', Gallagher [/bib_ref]. Postmortem situations related to MDAI, 5-Iodo-2-aminoindane (5-IAI), and 2-AI have been reported [bib_ref] ) toxicity: A brief overview and update, Corkery [/bib_ref] [bib_ref] A 3-year review of new psychoactive substances in casework, Elliott [/bib_ref] , the most illustrative example being a young woman who died of cardiac arrest, showing MDAI concentrations of 26.3 mg/L [91]. ## Synthetic cathinones Cathinone occurs naturally in the leaves of khat [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] , a plant usually found in East and Southern Africa, Southwest Arabian Peninsula, and Afghanistan [bib_ref] Khat (Catha edulis)-An updated review, Al-Hebshi [/bib_ref] [bib_ref] Chemical Pharmacology of Catha Edulis, Alles [/bib_ref]. More details on khat are presented in the plant-based NPS section. In 1975, cathinone, or 2-amino-1-phenylpropan-1-one, was identified and isolated from fresh khat leaves [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. This natural compound is a β-keto analogue of amphetamine, hence its amphetamine-like stimulant effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] shows the chemical structure of amphetamine and cathinone. ## Synthetic cathinones Cathinone occurs naturally in the leaves of khat [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] , a plant usually found in East and Southern Africa, Southwest Arabian Peninsula, and Afghanistan [bib_ref] Khat (Catha edulis)-An updated review, Al-Hebshi [/bib_ref] [bib_ref] Chemical Pharmacology of Catha Edulis, Alles [/bib_ref]. More details on khat are presented in the plant-based NPS section. In 1975, cathinone, or 2-amino-1-phenylpropan-1-one, was identified and isolated from fresh khat leaves [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. This natural compound is a β-keto analogue of amphetamine, hence its amphetamine-like stimulant effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] shows the chemical structure of amphetamine and cathinone. However, synthetic cathinones were first synthesized a few decades before, starting with methcathinone in 1928 [bib_ref] Synthetic homologues of d,l-ephedrine, Hyde [/bib_ref] , and mephedrone one year later. The structural resemblance of these compounds with classical amphetamines and their effects on the CNS brought the use of these compounds for clinical purposes to attention [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Meta chloro substituted-alpha-butylamino-propiophenones, Mehta [/bib_ref] , primarily as antidepressant and appetite suppressant drugs [bib_ref] Evaluation of pyrovalerone in chronically fatigued volunteers, Gardos [/bib_ref] [bib_ref] The use of diethylpropion in the treatment of obesity, Deramos [/bib_ref] [bib_ref] Bupropion hydrochloride ((+/−) alpha-t-butylamino-3-chloro-propiophenone HCl): A novel antidepressant agent, Soroko [/bib_ref]. It is also relevant to point out that only one synthetic cathinone is still available on the market for therapeutic purposes, bupropion, which is used as an antidepressant, coadjutant in smoking cessation therapy, and in the treatment of obesity [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Antidepressants for smoking cessation, Hughes [/bib_ref]. Despite their medicinal use, access to these compounds has been limited, considering their potential for abuse and dependence, and the adverse effects they might cause [bib_ref] Cathinone: An investigation of several N-alkyl and methylenedioxy-substituted analogs, Dal Cason [/bib_ref]. Methcathinone was the first compound used recreationally in the Soviet Union in the 1970s. Synthetic cathinones are commonly known as amphetamine derivatives not only because of their similar chemical structure to amphetamine [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] , but also concerning their physiological and behavioral effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. That said, in the beginning of the 21st century, specific cathinones started to appear in the market as legal substitutes for illegal substances, such as methamphetamine and MDMA [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref] , that were replaced by ephedrone and methylone-1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one, respectively [fig_ref] Figure 6: Chemical structures of methamphetamine, MDMA, ephedrone, and methylone [/fig_ref]. However, synthetic cathinones were first synthesized a few decades before, starting with methcathinone in 1928 [bib_ref] Synthetic homologues of d,l-ephedrine, Hyde [/bib_ref] , and mephedrone one year later. The structural resemblance of these compounds with classical amphetamines and their effects on the CNS brought the use of these compounds for clinical purposes to attention [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Meta chloro substituted-alpha-butylamino-propiophenones, Mehta [/bib_ref] , primarily as antidepressant and appetite suppressant drugs [bib_ref] Evaluation of pyrovalerone in chronically fatigued volunteers, Gardos [/bib_ref] [bib_ref] The use of diethylpropion in the treatment of obesity, Deramos [/bib_ref] [bib_ref] Bupropion hydrochloride ((+/−) alpha-t-butylamino-3-chloro-propiophenone HCl): A novel antidepressant agent, Soroko [/bib_ref]. It is also relevant to point out that only one synthetic cathinone is still available on the market for therapeutic purposes, bupropion, which is used as an antidepressant, coadjutant in smoking cessation therapy, and in the treatment of obesity [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Antidepressants for smoking cessation, Hughes [/bib_ref]. Despite their medicinal use, access to these compounds has been limited, considering their potential for abuse and dependence, and the adverse effects they might cause [bib_ref] Cathinone: An investigation of several N-alkyl and methylenedioxy-substituted analogs, Dal Cason [/bib_ref]. Methcathinone was the first compound used recreationally in the Soviet Union in the 1970s. Synthetic cathinones are commonly known as amphetamine derivatives not only because of their similar chemical structure to amphetamine [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] , but also concerning their physiological and behavioral effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. That said, in the beginning of the 21st century, specific cathinones started to appear in the market as legal substitutes for illegal substances, such as methamphetamine and MDMA [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref] , that were replaced by ephedrone and methylone-1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one, respectively [fig_ref] Figure 6: Chemical structures of methamphetamine, MDMA, ephedrone, and methylone [/fig_ref]. ## Synthetic cathinones Cathinone occurs naturally in the leaves of khat [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] , a plant usually found in East and Southern Africa, Southwest Arabian Peninsula, and Afghanistan [bib_ref] Khat (Catha edulis)-An updated review, Al-Hebshi [/bib_ref] [bib_ref] Chemical Pharmacology of Catha Edulis, Alles [/bib_ref]. More details on khat are presented in the plant-based NPS section. In 1975, cathinone, or 2-amino-1-phenylpropan-1-one, was identified and isolated from fresh khat leaves [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. This natural compound is a β-keto analogue of amphetamine, hence its amphetamine-like stimulant effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] shows the chemical structure of amphetamine and cathinone. However, synthetic cathinones were first synthesized a few decades before, starting with methcathinone in 1928 [bib_ref] Synthetic homologues of d,l-ephedrine, Hyde [/bib_ref] , and mephedrone one year later. The structural resemblance of these compounds with classical amphetamines and their effects on the CNS brought the use of these compounds for clinical purposes to attention [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Meta chloro substituted-alpha-butylamino-propiophenones, Mehta [/bib_ref] , primarily as antidepressant and appetite suppressant drugs [bib_ref] Evaluation of pyrovalerone in chronically fatigued volunteers, Gardos [/bib_ref] [bib_ref] The use of diethylpropion in the treatment of obesity, Deramos [/bib_ref] [bib_ref] Bupropion hydrochloride ((+/−) alpha-t-butylamino-3-chloro-propiophenone HCl): A novel antidepressant agent, Soroko [/bib_ref]. It is also relevant to point out that only one synthetic cathinone is still available on the market for therapeutic purposes, bupropion, which is used as an antidepressant, coadjutant in smoking cessation therapy, and in the treatment of obesity [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Antidepressants for smoking cessation, Hughes [/bib_ref]. Despite their medicinal use, access to these compounds has been limited, considering their potential for abuse and dependence, and the adverse effects they might cause [bib_ref] Cathinone: An investigation of several N-alkyl and methylenedioxy-substituted analogs, Dal Cason [/bib_ref]. Methcathinone was the first compound used recreationally in the Soviet Union in the 1970s. Synthetic cathinones are commonly known as amphetamine derivatives not only because of their similar chemical structure to amphetamine [fig_ref] Figure 5: Chemical structure of amphetamine and cathinone [/fig_ref] , but also concerning their physiological and behavioral effects [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. That said, in the beginning of the 21st century, specific cathinones started to appear in the market as legal substitutes for illegal substances, such as methamphetamine and MDMA [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref] , that were replaced by ephedrone and methylone-1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one, respectively [fig_ref] Figure 6: Chemical structures of methamphetamine, MDMA, ephedrone, and methylone [/fig_ref]. Innumerable cathinone derivatives can be synthesized by modifying the cathinone backbone structure. There are five different sites in which these modifications can occur [fig_ref] Figure 7: General chemical structure of synthetic cathinones [/fig_ref] [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. A total of 156 synthetic cathinones are currently monitored by the EU EWS, making it the second largest group of substances controlled by this organization. Innumerable cathinone derivatives can be synthesized by modifying the cathinone backbone structure. There are five different sites in which these modifications can occur [fig_ref] Figure 7: General chemical structure of synthetic cathinones [/fig_ref] [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. A total of 156 synthetic cathinones are currently monitored by the EU EWS, making it the second largest group of substances controlled by this organization. These compounds have gained outstanding popularity worldwide [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] due to their wide availability, relatively low prices, impressive marketing strategies, high purity compared to "street drugs", and popularization via the internet and in the so called smartshops. They are also known by the street names "Miaow Miaow", "M-Cat", "Msmack", "Drone", "Fert", or "Bubbles". Regulatory measures have been introduced to control these substances. However, new uncontrolled derivatives keep appearing on the market to replace those that have been subjected to legislation [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Some cathinone derivatives are internationally controlled. Cathinone and methcathinone are listed in Schedule I of the 1971 Single Convention on Psychotropic Substances, cathine in Schedule III, and pyrovalerone in Schedule IV. Methylone was the first synthetic cathinone reported to the EMCDDA in 2005, and it is part of the first generation of synthetic cathinones to be marketed as "legal high" [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. The legislative control of the first generation of synthetic cathinones and the demand for novel and legal substitutes increased the synthesis of new molecules. Mephedrone and MDPV appeared two years after methylone. Other compounds appeared ever since, for instance, the commonly found butylone, 4-methylethcathinone (4-MEC), and methedrone; and the less common α-pyrrolidinopentiophenone (α-PVP) and buphedrone. Some of these synthetic cathinones are shown in [fig_ref] Figure 8: Some of the most common synthetic cathinones [/fig_ref]. These compounds have gained outstanding popularity worldwide [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] due to their wide availability, relatively low prices, impressive marketing strategies, high purity compared to "street drugs", and popularization via the internet and in the so called smartshops. They are also known by the street names "Miaow Miaow", "M-Cat", "Msmack", "Drone", "Fert", or "Bubbles". Regulatory measures have been introduced to control these substances. However, new uncontrolled derivatives keep appearing on the market to replace those that have been subjected to legislation [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Some cathinone derivatives are internationally controlled. Cathinone and methcathinone are listed in Schedule I of the 1971 Single Convention on Psychotropic Substances, cathine in Schedule III, and pyrovalerone in Schedule IV. Methylone was the first synthetic cathinone reported to the EMCDDA in 2005, and it is part of the first generation of synthetic cathinones to be marketed as "legal high" [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. The legislative control of the first generation of synthetic cathinones and the demand for novel and legal substitutes increased the synthesis of new molecules. Mephedrone and MDPV appeared two years after methylone. Other compounds appeared ever since, for instance, the commonly found butylone, 4-methylethcathinone (4-MEC), and methedrone; and the less common α-pyrrolidinopentiophenone (α-PVP) and buphedrone. Some of these synthetic cathinones are shown in [fig_ref] Figure 8: Some of the most common synthetic cathinones [/fig_ref]. Innumerable cathinone derivatives can be synthesized by modifying the cathinone backbone structure. There are five different sites in which these modifications can occur [fig_ref] Figure 7: General chemical structure of synthetic cathinones [/fig_ref] [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. A total of 156 synthetic cathinones are currently monitored by the EU EWS, making it the second largest group of substances controlled by this organization. These compounds have gained outstanding popularity worldwide [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] due to their wide availability, relatively low prices, impressive marketing strategies, high purity compared to "street drugs", and popularization via the internet and in the so called smartshops. They are also known by the street names "Miaow Miaow", "M-Cat", "Msmack", "Drone", "Fert", or "Bubbles". Regulatory measures have been introduced to control these substances. However, new uncontrolled derivatives keep appearing on the market to replace those that have been subjected to legislation [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Some cathinone derivatives are internationally controlled. Cathinone and methcathinone are listed in Schedule I of the 1971 Single Convention on Psychotropic Substances, cathine in Schedule III, and pyrovalerone in Schedule IV. Methylone was the first synthetic cathinone reported to the EMCDDA in 2005, and it is part of the first generation of synthetic cathinones to be marketed as "legal high" [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. The legislative control of the first generation of synthetic cathinones and the demand for novel and legal substitutes increased the synthesis of new molecules. Mephedrone and MDPV appeared two years after methylone. Other compounds appeared ever since, for instance, the commonly found butylone, 4-methylethcathinone (4-MEC), and methedrone; and the less common α-pyrrolidinopentiophenone (α-PVP) and buphedrone. Some of these synthetic cathinones are shown in [fig_ref] Figure 8: Some of the most common synthetic cathinones [/fig_ref]. Some cathinone derivatives show only about half the stimulant potency of amphetamines [bib_ref] Patterns of use and toxicity of new para-halogenated substituted cathinones: 4-CMC (clephedrone),..., Grifell [/bib_ref] , and this leads to the consumption of higher doses or even re-dosing [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref]. These NPS are mainly consumed orally [bib_ref] Khat and synthetic cathinones: A review, Valente [/bib_ref] [bib_ref] Designer cathinones-An emerging class of novel recreational drugs, Zawilska [/bib_ref] , but nasal insufflation and inhalation, gingival and sublingual mucosal routes, intravenous, intramuscular, and subcutaneous injections [bib_ref] The effects and risks associated with synthetic cathinones use in humans, Karila [/bib_ref] [bib_ref] Abuse potential and toxicity of the synthetic cathinones (i.e., "Bath salts"), Riley [/bib_ref] , and rectal administrationmay be used as well. Cathinones can be consumed pure or in combination with another cathinone and/or with various other types of NPS, illicit drugs, medicines, anesthetic agents, and alcohol, to enhance the psychoactive effect [bib_ref] Khat and synthetic cathinones: A review, Valente [/bib_ref]. Concomitantly used with prescription drugs, some side effects can be reduced: BZPs help with anxiety; β-blockers are used with tachycardia; proton pump inhibitors help with stomach pain; and selective phosphodiesterase type 5 inhibitors increase libido and improve sexual performance [bib_ref] Designer cathinones-An emerging class of novel recreational drugs, Zawilska [/bib_ref] [bib_ref] Synthetic cathinones-Prevalence and motivations for use, Corkery [/bib_ref]. However, this concomitant consumption may be unintentional if the information about the content of drug is lacking. Doses vary largely from a few milligrams to a couple of grams depending on the substance that is being consumed. The route of administration is also important: when a synthetic cathinone is consumed by nasal insufflation, there is a quicker onset of the desired effects, so lower doses are needed in comparison to oral ingestion [bib_ref] Khat and synthetic cathinones: A review, Valente [/bib_ref]. A typical dose of mephedrone, for example, is 100-250 mg. On the other hand, MDPV is usually consumed in doses of 5-10 mg because it is more potent. In the United Kingdom, toxicity related to synthetic cathinones consumption increased from none to 600 cases from 2009 to 2010. Additionally in that year, the prevalence of consumption in Northern Ireland was about 2%. In France, MDPV and 4-Methylethcathinone (4-MEC) were two of the most consumed NPS. After consumption, these substances are first distributed to heavily irrigated organs such as brain, lungs, liver, and kidneys. It is in this phase that the first effects occur [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Synthetic cathinones show the ability to mediate the activity of monoamine transporters and receptors in the brain. These substances interact mainly with the serotonin receptor and transporters such as DAT, NET, and SERT. Synthetic cathinones block these receptors and transporters which, in turn, leads to an accumulation of monoamines such as dopamine, serotonin and noradrenaline in the synaptic cleft [bib_ref] Pharmacological profiles of aminoindanes, piperazines, and pipradrol derivatives, Simmler [/bib_ref] [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Effects of Synthetic Cathinones on Brain Neurotransmitters, Gołembiowska [/bib_ref] [bib_ref] The Designer Methcathinone Analogs, Mephedrone and Methylone, are Substrates for Monoamine Transporters..., Baumann [/bib_ref] [bib_ref] Bath salts components mephedrone and methylenedioxypyrovalerone (MDPV) act synergistically at the human..., Cameron [/bib_ref] [bib_ref] Inhibition of plasma membrane monoamine transporters by β-ketoamphetamines, Cozzi [/bib_ref] [bib_ref] The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat..., Nagai [/bib_ref]. Afterwards, distribution occurs throughout muscle, fat, and skin accounting for the redistribution of some drugs. Several factors condition this process, such as the ability of the substance to bind to plasma proteins and tissues, cardiac output, blood flow and capillary permeability, and local pH. In general, synthetic cathinones tend to bind poorly to plasma proteins [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Synthetic cathinones are subjected to phase I and II metabolism, mostly mediated through cytochrome P450. Notwithstanding, cathinones are also found non-metabolized in urine [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Khat and synthetic cathinones: A review, Valente [/bib_ref] [bib_ref] Cathinone derivatives: A review of their chemistry, Kelly [/bib_ref]. Chemical structure determines the main metabolic pathways for each substance [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. According to the EMCDDA, there are still few studies regarding ring substituted cathinones, pharmacokinetics and pharmacodynamics. Although, some studies with methcathinone in rats and humans indicate that N-demethylation and hydroxylation of keto groups and oxidation of alkyl groups in the ring are common pathways. Nevertheless, the fact that metabolism varies according to structures and that it is dependent on numerous other factors leads to an increased complexity, which results in a higher risk and unpredictable consequences when these substances are consumed simultaneously with others [bib_ref] Pharmacological profiles of aminoindanes, piperazines, and pipradrol derivatives, Simmler [/bib_ref] [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Effects of Synthetic Cathinones on Brain Neurotransmitters, Gołembiowska [/bib_ref] [bib_ref] The Designer Methcathinone Analogs, Mephedrone and Methylone, are Substrates for Monoamine Transporters..., Baumann [/bib_ref] [bib_ref] Bath salts components mephedrone and methylenedioxypyrovalerone (MDPV) act synergistically at the human..., Cameron [/bib_ref] [bib_ref] Inhibition of plasma membrane monoamine transporters by β-ketoamphetamines, Cozzi [/bib_ref] [bib_ref] The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat..., Nagai [/bib_ref]. Average half-life times have been reported for some of these substances, for instance between 1.5 and 4.3 h for cathinone [bib_ref] Pharmacokinetics of cathinone, cathine and norephedrine after the chewing of khat leaves, Toennes [/bib_ref] [bib_ref] Pharmacodynamics and pharmacokinetics of khat: A controlled study, Widler [/bib_ref]. Several pharmacodynamics and pharmacokinetics studies have been conducted using animal models. In phase I metabolism, the formation of hydroxyl metabolites is one of the most common and major metabolic pathways. N-dealkylation is the major metabolic pathway for cathinones presenting N-alkyl groups, whereas for methylenedioxyphenyl cathinones, some metabolites will result from demethylation followed by O-methylation. For Npyrrolidine cathinone derivatives, most metabolites will result from hydroxylation followed by dehydrogenation. The resultant compound is then further metabolized originating an aliphatic aldehyde [bib_ref] Metabolism of Synthetic Cathinones, Zaitsu [/bib_ref]. Glucuronidation is a common way of phase II metabolism. In 2015, Negreira et al. using microsomes of human liver concluded that methedrone is metabolized via N-demethylation, O-demethylation, and hydroxylation of the aliphatic side chain, as well as reduction of the β-ketone moiety [bib_ref] In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone..., Negreira [/bib_ref]. Kamata et al. reported metabolic pathways for methylone in both humans and rats, in which the β-ketone moiety reduction was not observed. The authors reported that, although a percentage of the dose was excreted unchanged, processes of N-demethylation and demethylenation, followed by O-methylation occurred. The most common metabolites in urine were 3 -hydroxy-4methoxymethcathinone and 4 -hydroxy-3 -methoxymethcathinone. Židková et al. reported for the same compound the metabolites 3 -hydroxy-4 -methoxycathinone and 4 -hydroxy-3 -methoxycathinone in rat urine. Concerning adverse effects, neurological, psychiatric, and cardiac systems are the most affected systems in the body. Other effects may also occur, which can lead to multiple organ failure and death [bib_ref] Designer cathinones-An emerging class of novel recreational drugs, Zawilska [/bib_ref] [bib_ref] The effects and risks associated with synthetic cathinones use in humans, Karila [/bib_ref] [bib_ref] Abuse potential and toxicity of the synthetic cathinones (i.e., "Bath salts"), Riley [/bib_ref] [bib_ref] Synthetic cathinones-Prevalence and motivations for use, Corkery [/bib_ref]. The most common neurological and psychiatric effects include agitation and anxiety, cognitive disorders, visual and auditory hallucinations, delusions, aggressive and erratic conduct, paranoia, psychosis, and seizures. Cases of stroke, encephalopathy, coma, and convulsions have also been reported [bib_ref] A 6-year review of new psychoactive substances at the Centre Antipoison Grand-Ouest..., Turcant [/bib_ref] [bib_ref] Identification of novel psychoactive substances 25B-NBOMe and 4-CMC in biologi-cal material using..., Wiergowski [/bib_ref] [bib_ref] Postmortem distribution of α-pyrrolidinobutiophenone in body fluids and solid tissues of a..., Wurita [/bib_ref] [bib_ref] Determination of "new psychoactive substances" in postmortem matrices using microwave derivatization and..., Margalho [/bib_ref] [bib_ref] Determination of Selected Cathinones in Blood by Solid-Phase Extraction and GC-MS, Antunes [/bib_ref]. Concerning cardiac effects, chest pain, hypertension, tachycardia, and cardiac arrest are documented [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Other body compartments can be affected, namely the gastrointestinal and hepatic, hematological, and respiratory systems [bib_ref] Designer cathinones-An emerging class of novel recreational drugs, Zawilska [/bib_ref] [bib_ref] The effects and risks associated with synthetic cathinones use in humans, Karila [/bib_ref] [bib_ref] Abuse potential and toxicity of the synthetic cathinones (i.e., "Bath salts"), Riley [/bib_ref] [bib_ref] Synthetic cathinones-Prevalence and motivations for use, Corkery [/bib_ref]. The repeated use of these substances at high doses may cause craving, dependence, tolerance, and withdrawal syndrome, and these effects have been documented [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref] [bib_ref] Khat and synthetic cathinones: A review, Valente [/bib_ref] [bib_ref] Designer cathinones-An emerging class of novel recreational drugs, Zawilska [/bib_ref] [bib_ref] The effects and risks associated with synthetic cathinones use in humans, Karila [/bib_ref]. There is little information concerning the pharmacology features and toxicity of these substances [bib_ref] Abuse liability and therapeutic potential of the Mitragyna speciosa (kratom) alkaloids mitragynine..., Hemby [/bib_ref] , which would be important to help clinicians dealing with intoxications. This is particularly relevant in acute intoxications, considering that the consumed substances are not known and several symptoms overlap with those induced by other situations or drugs of abuse (amphetamines, cocaine, and MDMA) [bib_ref] An updated review on synthetic cathinones, Soares [/bib_ref]. Because these substances are synthesized and sold in illicit markets, abusers do not precisely know what they are ingesting, often leading to unwanted effects, overdoses, and death [bib_ref] Cathinone derivatives: A review of their chemistry, Kelly [/bib_ref] [bib_ref] Abuse liability and therapeutic potential of the Mitragyna speciosa (kratom) alkaloids mitragynine..., Hemby [/bib_ref] [bib_ref] Analysis of NRG 'legal highs' in the UK: Identification and formation of..., Brandt [/bib_ref]. The first fatal case associated with the consumption of synthetic cathinones occurred in Sweden, in 2008, with mephedrone. Later, several other fatal cases concerning this substance appeared, although in most cases other drugs were also detected. The same happens with other synthetic cathinones, since they are usually consumed in drug cocktails. In 2019, Vignali et al. reported a fatality of a 27-year-old man, and 1-phenyl-2-(pyrrolidin-1-yl)hexan-1-one (α-PHP) was detected in several samples [bib_ref] Distribution of the synthetic cathinone αpyrrolidinohexiophenone in biological specimens, Vignali [/bib_ref]. In 2020, Adamowicz et al. reported a fatality of an 18-year-old man, in which 4-methyl-1-phenyl-2-(pyrrolidin-1-yl)pentan-1-one (α-PiHP) and 1-(4-chlorophenyl)-2-(methylamino)-1-propanone (4-CMC) were present [bib_ref] A case of intoxication with a new cathinone derivative α-PiHP-A presentation of..., Adamowicz [/bib_ref]. Lelièvre et al. also reported a fatality, in this case, a 39-year-old man, linked to the consumption of synthetic cathinones in 2020. Several of these compounds were detected in postmortem analysis, including 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) and 1-(4-methylphenyl)-2-(1-pyrrolidinyl)-1-hexanone (MPHP). ## Phenylethylamines Phenylethylamines are derivative compounds of phenylethylamine [fig_ref] Figure 9: Chemical structure of phenylethylamine [/fig_ref] , an organic compound and a natural monoamine alkaloid that acts as a stimulant in the CNS, and can act as an antimicrobial agent [bib_ref] Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical..., Pei [/bib_ref] [bib_ref] Pharmacologic amphetamines, physiologic neuromodulators, Berry [/bib_ref]. . J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW Phenylethylamines are derivative compounds of phenylethylamine ganic compound and a natural monoamine alkaloid that acts as a stimu and can act as an antimicrobial agent [bib_ref] Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical..., Pei [/bib_ref] [bib_ref] Pharmacologic amphetamines, physiologic neuromodulators, Berry [/bib_ref]. Phenylethylamines include many compounds capable of producin psychoactive effects. Some of the first phenylethylamines were synthesized early in the la methoxyphenyl)-N-methylpropan-2-amine, or P-methoxymethampheta for example, was first reported in 1938 [bib_ref] A preliminary behavioral investigation of PMMA, the 4-methoxy analog of methamphetamine, Glennon [/bib_ref]. Later, during the last two de century, other phenylethylamine derivatives were synthesized, featur from both 2C and D series. A major example is 2C-B, which derives from nylethylamine. Furthermore, in 1997, the studies of David Nichol that the hallucinogenic effects of compounds such as 2C-B or dimethoxy mine (DOB) were more potent than other natural hallucinogenic compou that point on, several other phenylethylamines were synthesized, such as substances [bib_ref] Some new psychoactive substances: Precursor chemicals and synthesis-driven end-products, Collins [/bib_ref]. 2C series substances include compounds with aromatic ring substitu (4-ethyl-2,5-dimethoxyphenyl) ethanamine] (2C-E) or 2C-B. D series su amphetamines with aromatic substitutions, such as [1-(4-iodo-2,5-dimeth pan-2-amine] (DOI) or [2,5-dimethoxy-4-chloroamphetamine] (DOC). NB tain an N-methoxybenzyl group, featuring compounds such as [formula] iodo phenyl)-N-[(2-methoxyphenyl)methyl]ethanamine] (25I-NBOMe) or [2- methoxyphenyl)-ı-[(2-methoxyphenyl)methyl]ethan-1-amine] (25C-NBO difurans encompass a range of compounds, such as [1-(4-bromofuro[2,3-f 8-yl)propan-2-amine] (bromo-dragonfly) and [2-(4-bromo-2,3,6,7-tetr f][1]benzofuran-8-yl)ethanamine] (2C-B-Fly) [/formula] , while other compounds b phenylethylamines' group are, for example, PMMA. The chemic few phenylethylamines are presented in [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Phenylethylamines include many compounds capable of producing stimulant and psychoactive effects. Some of the first phenylethylamines were synthesized early in the last century. 1-(4methoxyphenyl)-N-methylpropan-2-amine, or P-methoxymethamphetamine (PMMA), for example, was first reported in 1938 [bib_ref] A preliminary behavioral investigation of PMMA, the 4-methoxy analog of methamphetamine, Glennon [/bib_ref]. Later, during the last two decades of the 20th century, other phenylethylamine derivatives were synthesized, featuring compounds from both 2C and D series. A major example is 2C-B, which derives from a natural phenylethylamine. Furthermore, in 1997, the studies of David Nichols' team reported that the hallucinogenic effects of compounds such as 2C-B or dimethoxybromoamphetamine (DOB) were more potent than other natural hallucinogenic compounds [bib_ref] Dihydrobenzofuran analogues of hallucinogens. 4. Mescaline derivatives, Monte [/bib_ref]. From that point on, several other phenylethylamines were synthesized, such as benzodifuranyl substances [bib_ref] Some new psychoactive substances: Precursor chemicals and synthesis-driven end-products, Collins [/bib_ref]. 2C series substances include compounds with aromatic ring substitutions such as [2-. The chemical structures of a few phenylethylamines are presented in [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Several substances from the referred series are yet to fall under international control, being regulated only in a few countries. An important and relevant aspect of the consumption of phenylethylamines is shown in the NBOMe series compounds. These substances are often sold as LSD, but have higher toxicity. The consumption method varies with the specific type of phenylethylamines. Ingestion is the most common, but phenylethylamines can also be insufflated, or taken as pills or capsules. Substances such as the 2C series are found in other forms such as powders, liquids, and tablets. Phenylethylamines were the third most reported group of NPS worldwide (437 cases), right after synthetic cathinones and synthetic cannabinoids (684 and 665 cases, respectively). methoxyphenyl)-ı-[(2-methoxyphenyl)methyl]ethan-1-amine] (25C-NBOMe). The benzodifurans encompass a range of compounds, such as [1-(4-bromofuro[2,3-f] [1]benzofuran-8-yl)propan-2-amine] (bromo-dragonfly) and [2-(4-bromo-2,3,6,7-tetrahydrofuro[2,3f]benzofuran-8-yl)ethanamine] (2C-B-Fly), while other compounds belonging to the phenylethylamines' group are, for example, PMMA. The chemical structures of a few phenylethylamines are presented in [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Phenylethylamines are capable of inducing hallucinations, such as methamphetamine, cocaine, and other drugs, as they mediate activity of several amine receptors. In fact, a study from 1996 showed that DOI, a hallucinogen, acts as a partial agonist of 5-HT 2A receptors, involved in mediating the hallucinogenic effects of several drug compounds such as LSD [bib_ref] LSD and the phenethylamine hallucinogen DOI are potent partial agonists at 5-HT2A..., Marek [/bib_ref]. Compounds from the NBOMe series, such as 25I-NBOMe, have also been recognized as agonists of 5-HT 2A receptors. Phenethylamines derivatives can also be used for therapeutic purposes, since they can affect several systems, such as the serotoninergic, dopaminergic, and noradrenergic. Thus, these substances can be administered as appetite suppressants, vasoconstrictors, bronchodilators, or calcium channel blockers, and can be sold as stimulants, hallucinogens, anti-depressants, anorectics, hormones, neurotransmitters, or bronchodilators [bib_ref] Phenethylamine and related compounds in plants, Smith [/bib_ref]. Concerning adverse effects, compounds from the D series have been reported as potential vasoconstrictors and induce tachycardia, seizures, hallucinations, kidney failure, and others [bib_ref] Clinical toxicology of newer recreational drugs, Hill [/bib_ref]. As for the NBOMe series, adverse effects include cardiovascular problems, seizures, metabolic acidosis, and organ failure. As for the 2C series, various clinical pictures have been reported for different patients, such as serotonin toxicity or sympathomimetic syndrome [bib_ref] 2C or Not 2C: Phenethylamine Designer Drug Review, Dean [/bib_ref]. The symptoms are similar to other phenethylamines: hallucinations, euphoria, nausea, tachycardia, and respiratory depression, for example [bib_ref] 2C or Not 2C: Phenethylamine Designer Drug Review, Dean [/bib_ref] [bib_ref] New drugs of abuse in North America, Haroz [/bib_ref]. Little information is available concerning phenylethylamines' metabolism in humans. Several factors may influence the pharmacokinetic profile of these substances, namely the user's tolerance and toxicity or purity of the compounds [bib_ref] Metabolic pathways of 4-bromo-2,5-dimethoxyphenethylamine (2C-B): Analysis of phase I metabolism with hepatocytes..., Carmo [/bib_ref]. Theobald and Maurer [bib_ref] No Title Studies on the metabolism and toxicological detection of the designer..., Theobald [/bib_ref] identified the metabolites of 2C-E in rat urine by gas chromatography coupled to mass spectroscopy (GC-MS). The authors reported that the pathways responsible for the metabolization of the compounds are O-demethylation, Nacetylation, and hydroxylation, and identified metabolites such as N-acetyl-2C-E and trifluoroacetylated 2C-E. As for the NBOMe series compounds, a few studies in vivo and in vitro have indicated that the main metabolic pathways are O-demethylation, hydroxylation, and Ndemethoxybenzylation for different compounds within the series [bib_ref] Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and..., Caspar [/bib_ref] [bib_ref] Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of..., Nielsen [/bib_ref]. A few examples are described in a study performed by Šuláková et al., which identified the compounds hydroxy-25CN-NBOMe (isomer 2), dehydro-25CN-NBOMe, and O-demethyl-25CN-NBOMe in human liver microsomes. The consumption of phenylethylamines poses a serious threat to public health worldwide due to their toxicity. Serotoninergic syndrome, for instance, is one of the main dangers of phenethylamines consumption [bib_ref] New psychoactive substances (NPS) and serotonin syndrome onset: A systematic review, Schifano [/bib_ref]. Several case reports concerning hospitalizations or fatalities have been published in the literature. Bromo-dragonfly has been linked to several deaths in Scandinavia. Compounds from the 2C series such as [2-[2,5-dimethoxy-4-(propylsulfanyl)phenyl]ethan-1-amin] (2C-T-7) have been linked to three fatalities in which a drug cocktail was ingested [bib_ref] Postmortem identification and quantitation of 2,5-dimethoxy-4-npropylthiophenethylamine using GC-MSD and GC-NPD, Curtis [/bib_ref]. PMA and PMMA are the most common phenylethylamines to be associated with fatalities. Concerning specific cases, Stellpflug et al. analyzed samples from a non-fatal case of an 18-year-old woman that was admitted to the emergency department with seizures, tachycardia, agitation and hypertension associated with the consumption of 25I-NBOMe [bib_ref] 2-(4-Iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe): Clinical case with unique confirmatory testing, Stellpflug [/bib_ref]. Still concerning this compound, a fatal case of a 19-year-old man was also reported [bib_ref] Postmortem detection of 25I-NBOMe [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine] in fluids and tissues determined by high..., Poklis [/bib_ref]. Lastly, a recent example concerns a fatality of a 17-year-old man caused by the ingestion of 25B-NBOMe [bib_ref] A Case Report of Sudden Death and Insightful View of Google Trends..., Al-Imam [/bib_ref]. ## Tryptamines These substances are structurally similar to serotonin [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Tryptamines have high affinity for 5-HT receptors, and the induced hallucinations are mostly mediated by the 5-HT 2A receptor [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Some of these compounds also release dopamine, serotonin, and norepinephrine [bib_ref] Novel psychoactive substances (designer drugs): Overview and pharmacology of modulators of monoamine..., Liechti [/bib_ref]. Tryptamines occur naturally in plants, fungi, and animals [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. They play a very important role in the human body, since serotonin is a naturally-derived hormone involved in regulating the CNS, operating in the regulation of sleep, cognition, memory, temperature, and behavior [bib_ref] Recreational Use, Analysis and Toxicity of Tryptamines, Tittarelli [/bib_ref]. Ayahuasca is a hallucinogenic drink made from a plant called Banisteriospsis caapi, or an association with Psychotria viridis. [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref]. The leaves of the latter are rich in DMT, a tryptamine that also exists in other plants. This drink is normally used by the indigenous tribes of the Amazon jungle in religious rituals, and are also used to treat depression, anxiety, alcohol, tobacco, and drug addiction [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref]. Other substances of this group can be found in Psilocibo spp. fungi, a type of mushrooms widely distributed that has been used by indigenous people for sacred and therapeutic rituals in South America, India, Mexico, and Australia. These mushrooms contain two main compounds: psilocybin and psilocin [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref] [bib_ref] Psilocybin as a New Approach to Treat Depression and Anxiety in the..., Vargas [/bib_ref]. These two substances can be found in about 190 species of Psilocybe mushrooms [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Psilocybin and psilocin have similar properties to LSD, the reason why they became known worldwide as "magic mushrooms" [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Psilocybin as a New Approach to Treat Depression and Anxiety in the..., Vargas [/bib_ref]. Psilocybin is also known for its therapeutic potential, being effectively used in the treatment of resistant depression, as well as in the treatment of anxiety and depression in cancer patients [bib_ref] Psilocybin as a New Approach to Treat Depression and Anxiety in the..., Vargas [/bib_ref] [bib_ref] Psilocybin Therapeutic Research: The Present and Future Paradigm, Kargbo [/bib_ref]. Moreover, its therapeutic use has been suggested as effective and safe for treating alcohol dependence and for tobacco smoking cessation [bib_ref] Potential Therapeutic Effects of Psilocybin, Johnson [/bib_ref]. Another example of a natural hallucinogenic substance in this category is 5-hydroxy-N,N-dimethyltryptaline (5-OH-DMT), a positional isomer of psilocin. This compound and its derivative 5-methoxy-N,N-methyltryptaline (5-MeO-DMT) are the main psychoactive elements present in the venom of the American desert toad Bufo alvarius [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Lysergic acid amine or (8β)-9,10-didehydro-6-methyl-ergoline-8-carboxamide (LSA), an analogous to LSD, appears naturally in the seeds of Argyreia nervosa and Ipomoea violacea. The similarity in the structures of LSA and serotonin has triggered interest for the application of LSA in the therapy of mental disorders and treatment of alcoholism [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. These natural compounds attracted attention of drug developers, who, by changing their chemical structure, created NPS. Many of these new compounds are not objects of animal or human studies, their acute or long-term effects are not known, and neither are their possible interactions with other substances or toxicological risks; this lack of information poses a public health risk. [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. These drugs are sold worldwide through the internet, and are used as cheaper substitutes for classic hallucinogens [bib_ref] Novel psychoactive substances (designer drugs): Overview and pharmacology of modulators of monoamine..., Liechti [/bib_ref]. Serotonin and tryptamine are structurally very similar [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Tryptamines derive from the decarboxylation of tryptophan. This produces the indole ring typical of these compounds, which gives them the name of indolealkylamines [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Minor additions and modifications to the structure of indolealkylamines provide a virtually infinite supply of new tryptamine structures. (Supplementary Material) summarizes some of the most common synthetic and natural compounds of this family. The main characteristic of the structure is the indole moiety of the molecule, which is responsible for the hallucinogenic properties. The changes in positions R4 and R5 are those that tend to create most compounds [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Some of these compounds' chemical structures are represented in [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Tryptamines derive from the decarboxylation of tryptophan. This produces the indole ring typical of these compounds, which gives them the name of indolealkylamines [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Minor additions and modifications to the structure of indolealkylamines provide a virtually infinite supply of new tryptamine structures. (Supplementary Material) summarizes some of the most common synthetic and natural compounds of this family. The main characteristic of the structure is the indole moiety of the molecule, which is responsible for the hallucinogenic properties. The changes in positions R4 and R5 are those that tend to create most compounds [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Some of these compounds' chemical structures are represented in [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. There is currently little information on the metabolic pathways or enzymes involved in the metabolization process of tryptamines [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Concerning LSD, less than 1% is eliminated unchanged in urine, being widely metabolized. Five metabolites were observed in urine: 2-oxo-LSD, 2-oxo-3-hydroxy-LSD, N-demethyl-LSD, and 13-and 14-hydroxy-LSD glucuronide. Psilocybin is dephosphorylated, originating from psilocin, its active metabolite. It subsequently undergoes new metabolization to form 4-OH-IAA (acetic 4-hydroxyindol acid) and psilocin O-glucuronide [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Other tryptamine derivatives such as 5-MeO-DMT, 5-OH-DMT, and DMT undergo MAO-A mediated metabolization. The metabolic pathway of DMT catalyzed by MAO-A is not the only one. Some metabolites have been found in human urine and blood, such as N-methyltryptain (NMT), 2-methyl-1,2,3,4-tetra-hydro-beta-carbolin (2-MTHBC), and 1,2,3,4-tetra-hydro-beta-carbolin (THBC). These are, however, minor metabolites. DMT-Noxide (DMT-NO) is a biotransformation product by N-oxidation, N-demethylation, and cycling, after oral administration of ayahuasca [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. There is currently little information on the metabolic pathways or enzymes invo in the metabolization process of tryptamines [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Concerning LSD, less than 1% is inated unchanged in urine, being widely metabolized. Five metabolites were observ urine: 2-oxo-LSD, 2-oxo-3-hydroxy-LSD, N-demethyl-LSD, and 13-and 14-hydroxy glucuronide. Psilocybin is dephosphorylated, originating from psilocin, its active m olite. It subsequently undergoes new metabolization to form 4-OH-IAA (acetic 4-hy yindol acid) and psilocin O-glucuronide [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Other tryptamine derivatives such as 5-MeO-DMT, 5-OH-DMT, and DMT und MAO-A mediated metabolization. The metabolic pathway of DMT catalyzed by MA is not the only one. Some metabolites have been found in human urine and blood, su N-methyltryptain (NMT), 2-methyl-1,2,3,4-tetra-hydro-beta-carbolin (2-MTHBC), 1,2,3,4-tetra-hydro-beta-carbolin (THBC). These are, however, minor metabolites. D N-oxide (DMT-NO) is a biotransformation product by N-oxidation, N-demethylation When administered orally, substrates for MAO are rapidly metabolized, losing their hallucinogenic activity. This leads users of DMT and 5-MeO-DMT to also consume MAO inhibitors. Thus, ayahuasca also contains β-carbolines to obtain this effect. The metabolic pathway of MAO is reduced, increasing the psychoactive effect of these compounds [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. 5-Methoxy-diisopropyltryptamine (5-MeO-DiPT), a recent derivative of tryptamin, is metabolized by three distinct metabolic pathways. The first is O-demethylation; the second by hydroxylation and methylation; and the third by N-desalkylation [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. The easy access to these substances is a public health problem, as they are available on the internet, in nightclubs and raves, which seems to be a growing market. From insignificant to significant quantities, different substances are being sold worldwide, and usually, users do not know what they are buying [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Psilocybe mushrooms are usually consumed as infusions or eaten raw. The dose of psilocybin per mushroom varies, and so do the effects [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Tryptamines can be taken orally, intramuscularly, or intravenously, but they can also be smoked [bib_ref] Recreational Use, Analysis and Toxicity of Tryptamines, Tittarelli [/bib_ref]. These different routes of administration depend on the substance or on the consumer's preference [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. For instance, DMT, is not active orally, so it is usually smoked or administered intramuscularly, subcutaneously, or intravenously [bib_ref] Recreational Use, Analysis and Toxicity of Tryptamines, Tittarelli [/bib_ref]. The route of administration influences the onset and duration of action. Psilocybin has an onset of action of 20-40 min and a duration of 4-6 h when administered orally, but when administered intravenously, this onset is much faster (1-2 min), and the effects last up to 20 min [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. Tryptamines are known to have a short duration of action, which encourages repeated consumption. This results in higher consumption habits, increasing the risk of dependence [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. The popularity of synthetic tryptamines has been increasing due to their similarity to LSD at a reduced cost [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. According to the 2019 Global Drug Surveyreport, about 40% of drug users consume these substances, and tryptamines are the class with the highest increase in use. The most used substance in this family is LSD (17.5%), followed by psilocybin, or magic mushrooms (14.8%), DMT (4.2%), magic truffles (3.3%), and ayahuasca (1.1%) [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. The European Drugs Report states that the prevalence of LSD and magic mushrooms among young adults in the EU has values below 1% for both substances. However, there are exceptions for psilocybin mushrooms. Finland (2%), Estonia (1.6%), and the Netherlands (1.1%) are the countries with the highest prevalence of this drug. Concerning LSD, the country with the highest prevalence of use is Finland (2%), followed by Estonia (1.7%). As also happens with other NPS, the chemical constituents of the product are rarely fully described, or the information may be incorrect. Thus, there are risks associated with consumption and a possibility of overdose upon repeated administration [bib_ref] Toxicology and Analysis of Psychoactive Tryptamines, Malaca [/bib_ref]. The effects of tryptamines include visual hallucinations and mental and perception changes, such as hypersensitivity, perception of physical and temporal space, feeling of unreality and altered personality, as well as distortions, illusions, and auditory, visual, and sensory hallucinations [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Other neurological effects may also occur, such as clonus, ataxia, hyperreflexia, agitation, psychosis, delusions, paranoia, excitability, anterograde amnesia, cataplexy, and confusion. They can also induce tremors, seizures, and panic reactions (which can be called "bad trips"), depressive psychotic effects that usually occur in consumers with existing psychopathologies [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Hallucinations or changes in perception can appear days, months, or even years after tryptamine consumption ("flashbacks") [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Tryptamine users may exhibit physiological health changes, such as increased heart rate, hypertension, tachypnea, hyperthermia, rhabdomyolysis, renal failure, trismus, euphoria, anxiety, diarrhea, abdominal cramps, sweating, vomiting, palpitations, drowsiness, dysphoria and mydriasis [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. There is no evidence that the consumption might pose a life-threatening risk due to cardiovascular, renal, or hepatic changes, as these substances do not have much affinity for the receptors present in these systems [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. However, when the body is exposed to both tryptamines and IMAO, harmful results may occur. By prolonging the effects of tryptamines due to the inhibition of MAO, hyperserotonergic effects may occur, as well as serotonin toxicity. These phenomena occur due to the agonist behavior of tryptamines and IMAO [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Although not commonly directly associated with death, risks can trigger situations and induce behaviors that lead consumers to put themselves at risk [bib_ref] The hallucinogenic world of tryptamines: An updated review, Araújo [/bib_ref]. Some data reveal that the use of these drugs may be beneficial for the treatment of psychiatric conditions, emotional stress, tobacco dependence, and depression. LSD (34.1%), magic mushrooms (20.4%), ayahuasca (3.9%), DMT (2.4%), and 5-MeO-DMT (0.2%) are the most commonly used tryptamines in these cases . Bilhimer et al. [bib_ref] Acute Intoxication following Dimethyltryptamine Ingestion. Case Rep, Bilhimer [/bib_ref] reported a case of a 25-year-old man who was submitted to the emergency room after consuming a tea purchased on the internet, that contained ayahuasca. Laboratory findings indicated a Creatine Kinase concentration of 895 IU/L, a white blood cell count of 20 K/mm 3 , and positive urine immunoassay for amphetamines. A later analysis showed a concentration greater than 2000 ng/mL in the urine sample. Honyiglo et al. [bib_ref] Unpredictable Behavior Under the Influence of "Magic Mushrooms": A Case Report and..., Honyiglo [/bib_ref] reported a case of an 18-year-old French man who ingested magic mushrooms. The victim assumed unusually aggressive and excited behavior and had an increased urge to jump off the balcony, which he followed up with and died. The autopsy results determined that the cause of death was multiple trauma, secondary to an accidental high fall under the influence of psilocybin mushrooms. The toxicological analysis in postmortem samples revealed that consumption around 5 g of dried magic mushrooms had occurred a few hours before death. A total psilocin concentration of 2.230 ng/mL was found in urine samples, 60 and 67 ng/mL in peripheral and cardiac blood respectively, bile (3102 ng/mL), and vitreous humor (57 ng/mL). In addition to psilocin, other substances such as THC (1.34 ng/mL), OH-THC (0.53 ng/mL), and THC-COOH (1.88 ng/mL) were found. Attema-de Jonge reported the case of two young men, 25 and 32 years old, who showed signs of self-mutilation with knives after consuming psilocybin mushrooms. One of them also ingested cocaine, cannabis, and alcohol, which together with these tryptamines can increase the risk of dangerous behavior [bib_ref] Automutilation after consumption of hallucinogenic mushrooms, Attema-De Jonge [/bib_ref]. Sklerov et al. [bib_ref] A fatal intoxication following the ingestion of 5-methoxy-N,Ndimethyltryptamine in an ayahuasca preparation, Sklerov [/bib_ref] reported a case of a 25-year-old man, who was found dead the morning after the consumption of an extract of herbs containing β-carbolines and hallucinogenic tryptamines. Toxicological reports identified DMT, tetrahydroharmine, harmaline, harmine, and 5-MeO-DMT, which was the substance with the highest concentration. ## Synthetic cannabinoids Synthetic cannabinoids, also known as synthetic cannabinoid receptor agonists (SCRA), were laboratory created, with intention for use as therapeutic pharmaceuticals [bib_ref] Cannabinoids: From pot to lab, Papaseit [/bib_ref]. Several compounds were synthesized during the 1970s of the last century by the pharmaceutical industry and academic/research laboratories, and their names came from the person or institution responsible; for example, the JWH family was synthesized by John W. Huffman, while HU-210 was synthesized at the Hebrew University in Jerusalem. More than 450 SCRA were synthesized over the course of 20 years [bib_ref] Cannabinoids: From pot to lab, Papaseit [/bib_ref] , but their recreational use only emerged in the 2000s [bib_ref] Moving around the molecule: Relationship between chemical structure and in vivo activity..., Wiley [/bib_ref]. Nowadays, these substances are mostly classified according to their chemical structures [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref]. According to the EMCDDA, seven major groups of SCRA exist, namely naphthoylindoles, naphthylmethylindoles, naphthoylpyrroles, naphthylmethylindenes, phenylacetylindoles, cyclohexylphenols, and classical cannabinoids. SCRA classification is summarized in Supplementary Material . Structural changes are made to SCRA molecules forming new substances. Therefore, it is natural that alterations made on a single molecule can originate several others. Inside the aminoalkylindole group [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] , it is possible to find other subgroups, such naphthoylindoles, where changes made in the R group can originate from substances of the JWH family, for example JWH-018 and JWH-073, among others. On the other hand, if alterations are made on the central molecule of the benzoylindoles subgroup, it is possible to obtain RSC-4 or AM-694. In the same way, if the R or R1 is modified in the indole carboxamide subgroup, we can obtain APICA or even 5F-APICA substances. the benzoylindoles subgroup, it is possible to obtain RSC-4 or AM-694. In the same way, if the R or R1 is modified in the indole carboxamide subgroup, we can obtain APICA or even 5F-APICA substances. Information concerning the classification of SCRA can be found in the UNODC document about recommended methods to identify these compounds in seized materials. These compounds are sold illegally, usually added to plant materials (crushed leaves), wrapped in aluminium foil-herbal mixtures-but can also be sold in a solid or oily form, if in their pure state. Comparative to cannabis, these substances are essentially smoked, but oral use has been reported, and less commonly, injections. Recently, eliquids are also available [bib_ref] Vaping Synthetic Cannabinoids: A Novel Preclinical Model of E-Cigarette Use in Mice, Lefever [/bib_ref] [bib_ref] The dark cloud of recreational drugs and vaping, Blundell [/bib_ref] [bib_ref] E-cigarettes-An unintended illicit drug delivery system, Breitbarth [/bib_ref]. It is common, in prison surroundings, to soak papers or tissues with SCRA, and then smoke them with tobacco or vape them using electronic cigarettes [bib_ref] Synthetic cannabinoids: Epidemiology, pharmacodynamics, and clinical implications, Castaneto [/bib_ref]. Nowadays, it is possible to acquire these substances on online markets (darknet), through drug "dealers", or even by exchanging products with other consumers. An UNODC report from 2021 indicated that 29% of NPS monitored from 2010 to 2020 were SCRA. In the EU alone, 209 new SCRA were identified from 2018 to 2020. Such numbers pose a major concern in terms of legislation and public health, because most of these compounds are synthesized with impurities, contaminants, and adulterants [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref] [bib_ref] Comparison of "herbal highs" composition, Zuba [/bib_ref]. The same UNODC report showed that 16% of fatalities associated with NPS, were due to the use of SCRA. These substances are mainly used by adolescents and young adults, due to their interest in cannabis [bib_ref] Patterns of synthetic cannabinoid use in Australia, Barratt [/bib_ref] [bib_ref] College students and use of K2: An emerging drug of abuse in..., Hu [/bib_ref]. Chemically, SCRA are structurally different from THC. Still, they are lipophilic and nonpolar [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref]. Knowing pharmacokinetics and pharmacodynamics properties of SCRA is key to further understand the obtained results from case reports and others [bib_ref] Distribution of Synthetic Cannabinoids JWH-210, RCS-4 and ∆ 9-Tetrahydrocannabinol After Intravenous Administration..., Schaefer [/bib_ref]. SCRA produce physiological and psychotropic effects, varying in duration and severity. However, little is known about the pharmacology and toxicology of them and metabolites, with few studies made in humans [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref]. SCRA metabolism began to be under focus in the early 2000s [bib_ref] -naphthalenyl) methanone mesylate, a cannabinoid receptor agonist, Zhang [/bib_ref] [bib_ref] Characterization of rat liver microsomal metabolites of AM-630, a potent cannabinoid receptor..., Zhang [/bib_ref] [bib_ref] Identification of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the..., Zhang [/bib_ref]. In the same decade, Sobolevsky et al. were able to first identify JWH-018 metabolites in human urine samples of three individuals who confessed to have smoked at the very maximum 1 g of "Tropical Synergy" [bib_ref] Detection of JWH-018 metabolites in smoking mixture post-administration urine, Sobolevsky [/bib_ref]. Authors were able to detect 13 metabolites and concluded that these metabolites, even the phase I metabolites and phase II metabolites, are excreted through glucuronidation reactions [bib_ref] Detection of JWH-018 metabolites in smoking mixture post-administration urine, Sobolevsky [/bib_ref]. It is important to trace metabolites, since JWH-018 is not detected in urine [bib_ref] Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human..., Möller [/bib_ref]. Recently, Toennes et al. [bib_ref] Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in..., Toennes [/bib_ref] also studied the metabolism of JWH-018 in human serum samples. By using a 2 mg and 3 mg dose of this substance, the estimated half time of distribution is 0.40 h and 0.45 h, respectively. In addition, concentrations in JWH-018 reached their maximum blood Information concerning the classification of SCRA can be found in the UNODC document about recommended methods to identify these compounds in seized materials. These compounds are sold illegally, usually added to plant materials (crushed leaves), wrapped in aluminium foil-herbal mixtures-but can also be sold in a solid or oily form, if in their pure state. Comparative to cannabis, these substances are essentially smoked, but oral use has been reported, and less commonly, injections. Recently, e-liquids are also available [bib_ref] Vaping Synthetic Cannabinoids: A Novel Preclinical Model of E-Cigarette Use in Mice, Lefever [/bib_ref] [bib_ref] The dark cloud of recreational drugs and vaping, Blundell [/bib_ref] [bib_ref] E-cigarettes-An unintended illicit drug delivery system, Breitbarth [/bib_ref]. It is common, in prison surroundings, to soak papers or tissues with SCRA, and then smoke them with tobacco or vape them using electronic cigarettes [bib_ref] Synthetic cannabinoids: Epidemiology, pharmacodynamics, and clinical implications, Castaneto [/bib_ref]. Nowadays, it is possible to acquire these substances on online markets (darknet), through drug "dealers", or even by exchanging products with other consumers. An UNODC report from 2021 indicated that 29% of NPS monitored from 2010 to 2020 were SCRA. In the EU alone, 209 new SCRA were identified from 2018 to 2020. Such numbers pose a major concern in terms of legislation and public health, because most of these compounds are synthesized with impurities, contaminants, and adulterants [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref] [bib_ref] Comparison of "herbal highs" composition, Zuba [/bib_ref]. The same UNODC report showed that 16% of fatalities associated with NPS, were due to the use of SCRA. These substances are mainly used by adolescents and young adults, due to their interest in cannabis [bib_ref] Patterns of synthetic cannabinoid use in Australia, Barratt [/bib_ref] [bib_ref] College students and use of K2: An emerging drug of abuse in..., Hu [/bib_ref]. Chemically, SCRA are structurally different from THC. Still, they are lipophilic and nonpolar [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref]. Knowing pharmacokinetics and pharmacodynamics properties of SCRA is key to further understand the obtained results from case reports and others [bib_ref] Distribution of Synthetic Cannabinoids JWH-210, RCS-4 and ∆ 9-Tetrahydrocannabinol After Intravenous Administration..., Schaefer [/bib_ref]. SCRA produce physiological and psychotropic effects, varying in duration and severity. However, little is known about the pharmacology and toxicology of them and metabolites, with few studies made in humans [bib_ref] Spice' and other herbal blends: Harmless incense or cannabinoid designer drugs?, Auwärter [/bib_ref]. SCRA metabolism began to be under focus in the early 2000s [bib_ref] -naphthalenyl) methanone mesylate, a cannabinoid receptor agonist, Zhang [/bib_ref] [bib_ref] Characterization of rat liver microsomal metabolites of AM-630, a potent cannabinoid receptor..., Zhang [/bib_ref] [bib_ref] Identification of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the..., Zhang [/bib_ref]. In the same decade, Sobolevsky et al. were able to first identify JWH-018 metabolites in human urine samples of three individuals who confessed to have smoked at the very maximum 1 g of "Tropical Synergy" [bib_ref] Detection of JWH-018 metabolites in smoking mixture post-administration urine, Sobolevsky [/bib_ref]. Authors were able to detect 13 metabolites and concluded that these metabolites, even the phase I metabolites and phase II metabolites, are excreted through glucuronidation reactions [bib_ref] Detection of JWH-018 metabolites in smoking mixture post-administration urine, Sobolevsky [/bib_ref]. It is important to trace metabolites, since JWH-018 is not detected in urine [bib_ref] Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human..., Möller [/bib_ref]. Recently, Toennes et al. [bib_ref] Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in..., Toennes [/bib_ref] also studied the metabolism of JWH-018 in human serum samples. By using a 2 mg and 3 mg dose of this substance, the estimated half time of distribution is 0.40 h and 0.45 h, respectively. In addition, concentrations in JWH-018 reached their maximum blood concentration within minutes after intake. In this work the authors were able to detect JWH-018 pentanoic acid, JWH-018 N-(3-hydroxypentyl), JWH-018 N-(4-hydroxypentyl), and JWH-018 N-(5hydroxypentyl) metabolites in urine. Moreover, the t 1/2 of distribution of the studied metabolites ranges from 0.61 to 1.23 h, when consuming a 2 mg dose of JWH-018 and 0.58 to 1.10 h when 3 mg of the same substance was inhaled [bib_ref] Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in..., Toennes [/bib_ref]. In fact, glucuronidation is the preferred route for excretion of these substances, originating their metabolites [bib_ref] Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine, Moran [/bib_ref] [bib_ref] Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases, Chimalakonda [/bib_ref] [bib_ref] In vitro phase I metabolism of the synthetic cannabimimetic JWH-018, Wintermeyer [/bib_ref]. Furthermore, human uridine diphosphate-glucuronosyltransferase (UGT) is responsible for this mechanism [bib_ref] Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases, Chimalakonda [/bib_ref]. Kong et al. [bib_ref] Synthetic cannabinoids are substrates and inhibitors of multiple drug-metabolizing enzymes, Kong [/bib_ref] and Abbate et al. [bib_ref] The ongoing challenge of novel psychoactive drugs of abuse. Part I. Synthetic..., Abbate [/bib_ref] summarize the metabolism of JWH-018 and Chimalakonda et al. [bib_ref] Cytochrome P450-Mediated Oxidative Metabolism of Abused Synthetic Cannabinoids Found in K2/Spice: Identification..., Chimalakonda [/bib_ref] describe the metabolism of JWH-018, as well as AM-2201 with its main metabolites. In a study about the metabolism of 5F-AB-P7AICA, Giorgetti et al. [bib_ref] Detection and phase I metabolism of the 7-azaindole-derived synthetic cannabinoid 5F-AB-P7AICA including..., Giorgetti [/bib_ref] proposed a metabolic pathway. After self and controlled administration, it was possible to detect a peak of the compound in urine samples, suggesting that this compound can be monitored without considering its metabolites. Moreover, 10 metabolites were confirmed in urine. The parent compound originated from hydroxylated, dehydrogenated compounds, as well as the metabolites originated from hydrolytic defluorination and amide hydrolysis [bib_ref] Detection and phase I metabolism of the 7-azaindole-derived synthetic cannabinoid 5F-AB-P7AICA including..., Giorgetti [/bib_ref]. Cannabinoids and SCRA exert their effects on the endocannabinoid system, mainly in the cannabinoid receptors 1 and 2 (CB 1 and CB 2 ), yet with higher binding affinities [bib_ref] Pharmacological and Toxicological Effects of Synthetic Cannabinoids and Their Metabolites, Tai [/bib_ref]. Data are limited about adverse effects and intoxications associated with the consumption of SCRA, because of their constant structural changes, which culminates in different potencies, efficacies, and duration of action. Nonetheless, studies were able to demonstrate effects of such compounds, for instance, seizures, tachyarrhythmia, anxiety, hallucinations and confusion, mydriasis, drowsiness, hypo and hypertension, and vomiting. [bib_ref] Severe Toxicity Following Synthetic Cannabinoid Ingestion, Lapoint [/bib_ref] [bib_ref] New Synthetic Cannabinoids Intoxications in Italy: Clinical Identification and Analytical Confirmation of..., Locatelli [/bib_ref]. Headaches, slowed speech, sweating, aggressiveness, lethargy and slowed speech have also been reported for other SCRA [bib_ref] Clinical Presentation of Intoxication Due to Synthetic Cannabinoids, Cohen [/bib_ref]. Cardiovascular effects as arrhythmias, cardiac arrest, cardiomyopathy, coronary thrombosis, acute myocardial infarction, and vasculitis have been reported [bib_ref] A Characterization of Synthetic Cannabinoid Exposures Reported to the National Poison Data..., Hoyte [/bib_ref] [bib_ref] A systematic review of adverse events arising from the use of synthetic..., Tait [/bib_ref] [bib_ref] Synthetic Cannabinoids-"Spice" Can Induce a Psychosis: A Brief Review, Yeruva [/bib_ref] [bib_ref] Cardiovascular effects of marijuana and synthetic cannabinoids: The good, the bad, and..., Pacher [/bib_ref]. A 34-year-old male died after consuming 5-Fluoro-ADB and was submitted to autopsy. The target compound was found in various matrices, including adipose tissue and heart muscle, but not in urine or blood fluids. The highest concentration was reported in adipose tissue, 7.95 ng/mL, followed by stomach and brain, with 3.18 ng/mL and 1.90 ng/mL, respectively. Less than 0.5 ng/mL was reported in lung, liver, and skeletal muscle. After inhalation, and loss of consciousness, death was due to cardiopulmonary arrest. A year later, the same authors were able to perform a trial identification of 5-fluro-ADB-PINACA and MAB-CHMINACA, except for urine, in which MAB-CHMINACA was not detected. Concentrations found ranged from 6.05 ng/mL to 156 ng/mL, the highest concentration was detected in liver tissue, followed by kidney and pancreas [bib_ref] Postmortem distribution of MAB-CHMINACA in body fluids and solid tissues of a..., Hasegawa [/bib_ref]. Minakata et al.determined 6 SCRA (AB-PINACA, AB-FUBINACA, AB-CHMINACA, MAB-CHMINACA, and 5F-AMB NS 5F-ADB) in the urine of three different cadavers. AB-PINACA and AB-FUBINACA were found in victim 1, with concentrations of 23 pg/mL and 10 pg/mL, respectively. Moreover, 239 pg/mL of AB-CHMINACA was detected in victim 2 and 229 pg/mL MAB-CHMINACA in victim 3. 5F-ADB was found both in victim 2 and 3, with concentrations of 19 pg/mL, each. Three fatal cases involving 5F-ADB, 5F-PB-22, and AB-CHMINACA were reported by Angerer et al. [bib_ref] Three fatalities associated with the synthetic cannabinoids 5F-ADB, 5F-PB-22, and AB-CHMINACA, Angerer [/bib_ref]. In this case, it is widely believed that death was due to consumption of these substances, as well as concomitant use of ethanol in two of the cases. In the other, toxic effects of antidepressants and 5F-ADB are thought to be the cause of death [bib_ref] Three fatalities associated with the synthetic cannabinoids 5F-ADB, 5F-PB-22, and AB-CHMINACA, Angerer [/bib_ref]. Another study hypothesized that SCRA consumption could be related to diabetic ketoacidosis and ultimately death [bib_ref] Death due to diabetic ketoacidosis: Induction by the consumption of synthetic cannabinoids?, Hess [/bib_ref]. Blood samples were collected and AB-CHMINACA, AB-FUBINACA, AM-2201, 5F-AMB, 5F-APINACA, EAM-2201, JWH-018, JWH-122, MAM-2201, STS135, and THJ 2201 were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The 25-year-old victim had a drug consumption history. Apart from the mentioned SCRA and medicinal drugs for diabetes (insulin), no other substances were detected. Allegedly, the cause of death was due to SCRA consumption and the lack of consumption of insulin, since high levels of glycosylated hemoglobin and acetone were detected in blood [bib_ref] Death due to diabetic ketoacidosis: Induction by the consumption of synthetic cannabinoids?, Hess [/bib_ref]. ## Phencyclidine analogs Phencyclidine-like substances belong to the arylcyclohexamines family, a group of chemicals considered to be dissociative hallucinogens, which have structures similar to those of PCP and ketamine [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. the lack of consumption of insulin, since high levels of glycosylated hemoglobin and acetone were detected in blood [bib_ref] Death due to diabetic ketoacidosis: Induction by the consumption of synthetic cannabinoids?, Hess [/bib_ref]. ## Phencyclidine analogs Phencyclidine-like substances belong to the arylcyclohexamines family, a group of chemicals considered to be dissociative hallucinogens, which have structures similar to those of PCP and ketamine [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. PCP was first synthesized in 1956 as an intravenous anaesthetic for humans, being approved by the FDA in 1957 and sold until the late 1960s. However, it was withdrawn from the market due to adverse psychological effects that were often disturbing and sometimes severe and prolonged [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref] [bib_ref] Ketamine and Other Phencyclidine Analogues: A Review of Their Use as Drugs..., Vargas [/bib_ref]. It was reintroduced as an animal tranquillizer in the late 1960s [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref]. The first report of recreational use of the drug was in the late 1960s in San Francisco under the name of "peace pill". Even though PCP was the first synthesized arylcyclohexylamine, other compounds had been reported before. For example, 1-(1-phenylcyclohexylamine)amine (PCA) was first stated in 1907, in 1953 Nethyl-1-phenylcyclohexylamine (PCE) was investigated, and 1-(1-Phenylcyclohexyl) morpholine (PCMo) in 1954 [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. Approximately 14 analogues were reported between 1960 and 1990. Since then, many more have been published . Still, the use of three compounds was prominent, considered to be in the first generation of phenylcyclohexyl analogues: PCE, TCP, and 1-(1-Phenylcyclohexyl) pyrrolidine (PCPy) [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] DARK Classics in Chemical Neuroscience: Phencyclidine (PCP), Bertron [/bib_ref]. The use of PCP has declined since the 1980s. Although it remains popular and its consumption is increasing, especially for nightclub attendees, it essentially is confined to the United States and Canada, with occasional reports in the European Union [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref]. According to the Drug Abuse Warning Network, in 2011, there were 75,538 emergency department visits, most of which were due to combinations with other drugs, such as marijuana, cocaine, painkillers, and anxiolytics. Two deaths from PCP were reported in 2012, according to the American Association of Poison Control Centers [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref]. To date, there is no international protocol for controlling these drugs. Nonetheless, there are specific European countries (e.g., France or Germany) with laws that banned PCP by the narcotics act [bib_ref] Beyond Ketamine and Phencyclidine: Analytically Confirmed Use of Multiple Novel Arylcyclohexylamines, Thornton [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. Since phencyclidine-type substances are most common PCP was first synthesized in 1956 as an intravenous anaesthetic for humans, being approved by the FDA in 1957 and sold until the late 1960s. However, it was withdrawn from the market due to adverse psychological effects that were often disturbing and sometimes severe and prolonged [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref] [bib_ref] Ketamine and Other Phencyclidine Analogues: A Review of Their Use as Drugs..., Vargas [/bib_ref]. It was reintroduced as an animal tranquillizer in the late 1960s [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref]. The first report of recreational use of the drug was in the late 1960s in San Francisco under the name of "peace pill". Even though PCP was the first synthesized arylcyclohexylamine, other compounds had been reported before. For example, 1-(1-phenylcyclohexylamine)amine (PCA) was first stated in 1907, in 1953 N-ethyl-1-phenylcyclohexylamine (PCE) was investigated, and 1-(1-Phenylcyclohexyl) morpholine (PCMo) in 1954 [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. Approximately 14 analogues were reported between 1960 and 1990. Since then, many more have been published . Still, the use of three compounds was prominent, considered to be in the first generation of phenylcyclohexyl analogues: PCE, TCP, and 1-(1-Phenylcyclohexyl) pyrrolidine (PCPy) [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] DARK Classics in Chemical Neuroscience: Phencyclidine (PCP), Bertron [/bib_ref]. The use of PCP has declined since the 1980s. Although it remains popular and its consumption is increasing, especially for nightclub attendees, it essentially is confined to the United States and Canada, with occasional reports in the European Union [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref]. According to the Drug Abuse Warning Network, in 2011, there were 75,538 emergency department visits, most of which were due to combinations with other drugs, such as marijuana, cocaine, painkillers, and anxiolytics. Two deaths from PCP were reported in 2012, according to the American Association of Poison Control Centers [bib_ref] Linking phencyclidine intoxication to the tryptophan-kynurenine pathway: Therapeutic implications for schizophrenia, Fujigaki [/bib_ref]. To date, there is no international protocol for controlling these drugs. Nonetheless, there are specific European countries (e.g., France or Germany) with laws that banned PCP by the narcotics act [bib_ref] Beyond Ketamine and Phencyclidine: Analytically Confirmed Use of Multiple Novel Arylcyclohexylamines, Thornton [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. Since phencyclidine-type substances are most common in the United States, PCE, PHP, PCPY, and TCP are controlled in Schedule I of the 1971 Convention, while PCP, PCC, and PCA are controlled in Schedule II of the 1978 Convention [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. A critical review on ketamine and other phencyclidine analogues was published by Vargas et al. [bib_ref] Ketamine and Other Phencyclidine Analogues: A Review of Their Use as Drugs..., Vargas [/bib_ref]. Ketamine was created in 1962, and subsequently commercialized in 1969. There is no evidence of long-term neurotoxicity or prolonged unfavorable psychological effects when used in a controlled clinical setting. This analogue is popular worldwide and has several therapeutic applications, including general anaesthesia, analgesia, depression, psychiatric treatment, and veterinary use [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref] [bib_ref] DARK Classics in Chemical Neuroscience: Phencyclidine (PCP), Bertron [/bib_ref]. In 2019, the FDA approved a new nasal spray medication for the treatment of depression, containing esketamine, an S-enantiomer of ketamine. The first report of ketamine use for its psychedelic effects was in 1971. Illicit ketamine is commonly named "Special K"," Ket", "vitamin K", and is often found as a white crystalline powder or pharmaceutically packaged injectable solution and is usually inflated, injected, or less commonly, consumed orally [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. It is often adulterated by the addition of caffeine, MDMA, heroin, or cocaine. Supplementary Material shows a study with 126 patients without any fatal cases, but with different levels of toxicity. Ketamine is highly lipid-soluble; therefore, distribution to the CNS is enabled. It has a half-life of 3 h, binding affinity of about 10-30% to proteins and its oral bioavailability is 17%. The onset of action of ketamine and methoxetamine (a new derivative) is rapid following intravenous and intramuscular administration, with effects apparent after 1 min and 5 min, respectively. After nasal insufflation, the onset of action is up to 30 min, being up to 30-90 min. for methoxetamine. Ketamine is metabolized primarily by the CYP2B6 isoenzyme, with contributions from CYP3A4 and CYP2C9. Ketamine's main metabolic pathway is N-demethylation to norketamine, its primary metabolite, which has one-third the potency of ketamine. Norketamine undergoes hydroxylation on the hexanone ring, and subsequent glucuronidation to more water-soluble metabolites that are excreted in the urine and also in the bile. The increasing reports of urotoxicity associated with the chronic use of ketamine in high doses have led to the emergence of methoxetamine [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. Methoxetamine [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] is a derivative of ketamine and has similar effects; therefore, the potential for abuse is also high. It has been suggested that methoxetamine may share the same neuropharmacological properties as NMDA antagonist and dopamine reuptake inhibitor [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. in the United States, PCE, PHP, PCPY, and TCP are controlled in Schedule I of the 1 Convention, while PCP, PCC, and PCA are controlled in Schedule II of the 1978 C vention [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. A critical review on ketamine and other phencyclidine analogues was published Vargas et al. [bib_ref] Ketamine and Other Phencyclidine Analogues: A Review of Their Use as Drugs..., Vargas [/bib_ref]. Ketamine was created in 1962, and subsequently commercialized in 1969. There i evidence of long-term neurotoxicity or prolonged unfavorable psychological effects w used in a controlled clinical setting. This analogue is popular worldwide and has sev therapeutic applications, including general anaesthesia, analgesia, depression, psychia treatment, and veterinary use [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Designer drugs: A medicinal chemistry perspective, Carroll [/bib_ref] [bib_ref] DARK Classics in Chemical Neuroscience: Phencyclidine (PCP), Bertron [/bib_ref]. In 2019, the FDA approved a new sal spray medication for the treatment of depression, containing esketamine, an S-ena omer of ketamine. The first report of ketamine use for its psychedelic effects was in 1971. Illicit ketam is commonly named "Special K"," Ket", "vitamin K", and is often found as a white c talline powder or pharmaceutically packaged injectable solution and is usually infla injected, or less commonly, consumed orally [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref]. It is often adulterated by the addi of caffeine, MDMA, heroin, or cocaine. Supplementary Material show study with 126 patients without any fatal cases, but with different levels of toxicity [2 Ketamine is highly lipid-soluble; therefore, distribution to the CNS is enabled. It h half-life of 3 h, binding affinity of about 10-30% to proteins and its oral bioavailabili 17%. The onset of action of ketamine and methoxetamine (a new derivative) is ra following intravenous and intramuscular administration, with effects apparent aft min and 5 min, respectively. After nasal insufflation, the onset of action is up to 30 m being up to 30-90 min. for methoxetamine. Ketamine is metabolized primarily by the CYP2B6 isoenzyme, with contribut from CYP3A4 and CYP2C9. Ketamine's main metabolic pathway is N-demethylatio norketamine, its primary metabolite, which has one-third the potency of ketamine. N ketamine undergoes hydroxylation on the hexanone ring, and subsequent glucuron tion to more water-soluble metabolites that are excreted in the urine and also in the. The increasing reports of urotoxicity associated with the chronic use of ketamin high doses have led to the emergence of methoxetamine [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. Methoxetamine [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] is a derivative of ketamine and has similar effects; th fore, the potential for abuse is also high. It has been suggested that methoxetamine share the same neuropharmacological properties as NMDA antagonist and dopam reuptake inhibitor [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. Methoxetamine first appeared in 2010 and was widely marketed by "major sto on the internet, which led to several reports of hospitalization [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. Several ro of administration are possible, including intramuscular, oral, inhalation, rectal, or in venous. So far, the metabolism of methoxetamine is not completely known, but it app that CYP2B6 and CYP3A4 are involved. The primary metabolite, normethoxetamine sults from N-deethylation and undergoes hydroxylation and glucuronidation to form more water-soluble hydroxynormethoxetamine glucuronide. These metabolites Methoxetamine first appeared in 2010 and was widely marketed by "major stores" on the internet, which led to several reports of hospitalization [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref]. Several routes of administration are possible, including intramuscular, oral, inhalation, rectal, or intravenous. So far, the metabolism of methoxetamine is not completely known, but it appears that CYP2B6 and CYP3A4 are involved. The primary metabolite, normethoxetamine, results from N-deethylation and undergoes hydroxylation and glucuronidation to form the more water-soluble hydroxynormethoxetamine glucuronide. These metabolites are consistent with those found in urine samples from analytically confirmed methoxetamine cases. Both ketamine and methoxetamine have similar effects, considered positive effects such as euphoria and having a sense of calm and serenity, neutral effects such as dis-tortion or loss of sensory perception, and harmful effects such as severe dissociation, depersonalization, loss of consciousness, nausea, and vomiting [bib_ref] Methoxetamine (MXE)-A Phenomenological Study of Experiences Induced by a "Legal High" from..., Kjellgren [/bib_ref] [bib_ref] The evolving high: New designer drugs of abuse, Pourmand [/bib_ref]. According to Botanas et al. [bib_ref] R (−)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side..., Botanas [/bib_ref] , MXE and enantiomers have antidepressant effects via glutamatergic and serotonergic mechanisms, as also happens with ketamine,. The synthesis of 4-MeO-PCP dates back to 1965 as part of a systematic search for PCP analogues with potential clinical use [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. The first signs of underground 4-MeO-PCP experimentation began to surface in 1999, but it was not until about 2008 that it arrived in online shops as one of the first dissociative drugs [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref]. 4-MeO-PCP is available as powder or tablet, is active via the oral and parenteral routes, and is reported to induce dissociative effects, but with substantially reduced potency relative to PCP and 3-MeO-PCP [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. They also present appreciable affinities for the serotonin transporter and high affinities for sigma receptors [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. The dosage of 4-MeO-PCP is much higher compared to others, as the usual oral dose can be up to 250 mg. This difference in drug activity between isomers can be responsible for acute and severe intoxication in case of confusion by addicts [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. 3-MeO-PCP became available online as a research chemical in 2011 [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. It is known that 3-MeO-PCP is a more potent NMDA receptor antagonist than PCP, presenting almost three times the affinity [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref]. They also present appreciable affinities for the serotonin transporter and high affinities for sigma receptors [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. 3-MeO-PCP is available as powder or tablets and is commonly administered orally but can also be injected, snorted, or smoked. Sublingual, intramuscular, and rectal administration have also been reported [bib_ref] From PCP to MXE: A comprehensive review of the non-medical use of..., Morris [/bib_ref] [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref] [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref] [bib_ref] Intoxication with 3-MeO-PCP alone, Berar [/bib_ref]. Elimination has been estimated to be approximately 11 h [bib_ref] Phencyclidine-based new psychoactive substances, Wallach [/bib_ref]. Abuse doses are usually between 5 to 20 mg. Duration of effects has been reported to be of approximately 4.5 h, with onset within 30 min after oral ingestion and a peak at approximately 2 h [bib_ref] A non-fatal intoxication and seven deaths involving the dissociative drug 3-MeO-PCP, Johansson [/bib_ref]. It produces many adverse effects, including psychosis, confusion, violent behavior, hypertension, tachycardia, suicidal impulses, and coma to death [bib_ref] Murdered while under the influence of 3-MeO-PCP, Kintz [/bib_ref]. ## Synthetic opioids Opioids cause anesthetic and depressant effects; however, their recreational use has been of public health concern. According to the UNODC report from 2013, up until this date, the only opioid in the spotlight among NPS was O-desmethyltramadol (the main active metabolite of tramadol) that was consumed together with kratom [bib_ref] Automated high-throughput analysis of tramadol and O-desmethyltramadol in dried blood spots, Luginbühl [/bib_ref]. Novel synthetic opioids, or simply synthetic opioids, include fentanyl and analogues, but compounds such as tramadol, methadone, pethidine, ketobemidone, levorphanol, dextromoramide, dipipanone, and others are also included in this category. However, the majority of these are already under regulation policies, which is not the case of most abused substances. Amongst recent fentanyl analogues, the WHO highlights acetylfentanyl, butyrylfentanyl (or butyrylfentanyl), acryloylfentanyl (or acrylfentanyl), carfentanil, furanylfentanyl, and ocfentanil [bib_ref] Acute Intoxications and Fatalities from Illicit Fentanyl and Analogues: An Update, Pichini [/bib_ref]. Most common non-medical abused substances include fentanyl analogues, and benzamide opioids, such as AH-7921 and U-47700, and others such as MT-45 and W-18, which are piperazine opioids [bib_ref] Global strategy for New Psychoactive Substances: An update, Vicknasingam [/bib_ref]. Sometimes, these substances can be found in counterfeit opioid pills, or other pills that imitate benzodiazepines, and heroin. Fentanyl was produced in the 1950s [bib_ref] Abuse of fentanyl: An emerging problem to face, Kuczyńska [/bib_ref] , aiming at finding molecules with good therapeutic properties, but with less undesired side effects [bib_ref] The Fentanyl Story, Stanley [/bib_ref]. Firstly, it was established to be medically used, as it is still today, for chronic pain treatment and intraoperative analgesia [bib_ref] Designer drugs: Mechanism of action and adverse effects, Luethi [/bib_ref] [bib_ref] The Fentanyl Story, Stanley [/bib_ref]. U-47700 and AH-7921 are isomers and were synthetized in the 1970s with the purpose to replace classic opioids, but their production was abandoned due to addictive effects. Although it was first produced in the 1970s, MT-45, or IC-6, first appeared on an EMCDDA report only in 2013 [bib_ref] Synthesis and structure-activity relationships of 1-substituted 4-(1,2-diphenylethyl)piperazine derivatives having narcotic agonist and..., Natsuka [/bib_ref] [bib_ref] 1-Substituted 4-(1,2-diphenylethyl)piperazine derivatives and their analgesic activities. 1, Natsuka [/bib_ref]. Novel synthetic opioids are responsible for the worldwide opioid crisis, with special incidence in the United States [bib_ref] Novel Synthetic Opioids: An Opioid Epidemic Within an Opioid Epidemic, Lucyk [/bib_ref] [bib_ref] Increases in Drug and Opioid-Involved Overdose Deaths-United States, Rudd [/bib_ref]. In fact, from 2009 to 2019, a total of 77 NPS with opioid effects were reported by the UNODC. This phenomenon poses a challenge to public health authorities, as well as legislative authorities to avoid the dissemination, dependence, overdoses, and inappropriate use of these substances. Synthetic opioids are basically categorized into fentanyl and analogues, and nonfentanyl structured compounds.The former may be further divided into pharmaceutical fentanyls, which include fentanyl, for example, alfentanil and sufentanyl; and non-pharmaceutical fentanyls, which include fentanyl analogues and designer fentanyls, for instance ocfentanyl and bythyrfentanyl [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. Fentanyl, an analgesic analogue of phenopiperidine, is presented on [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. responsible for the worldwide opioid crisis, with special incidence in the United State [bib_ref] Novel Synthetic Opioids: An Opioid Epidemic Within an Opioid Epidemic, Lucyk [/bib_ref] [bib_ref] Increases in Drug and Opioid-Involved Overdose Deaths-United States, Rudd [/bib_ref]. In fact, from 2009 to 2019, a total of 77 NPS with opioid effects were reported by the UNODC. This phenomenon poses a challenge to public health authorities, a well as legislative authorities to avoid the dissemination, dependence, overdoses, and in appropriate use of these substances. Synthetic opioids are basically categorized into fentanyl and analogues, and non-fen tanyl structured compounds.The former may be further divided into pharmaceutica fentanyls, which include fentanyl, for example, alfentanil and sufentanyl; and non-phar maceutical fentanyls, which include fentanyl analogues and designer fentanyls, for in stance ocfentanyl and bythyrfentanyl [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. Fentanyl, an analgesic analogue of phe nopiperidine, is presented on [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Fentanyl analogues [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] followed fentanyl synthesis and were implemented in veterinary medicine, namely sufentanil, alfentanil, remifentanil, and carfentanil. Othe analogues include acetylfentanyl, acryloylfentanyl, α-methylfentanyl, 3-methylfentanyl furanylfentanyl, cyclopentylfentanyl, and ocfentanil. However, carfentanil is th most used recreationally. Fentanyl analogues [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] followed fentanyl synthesis and were implemented in veterinary medicine, namely sufentanil, alfentanil, remifentanil, and carfentanil. Other analogues include acetylfentanyl, acryloylfentanyl, α-methylfentanyl, 3-methylfentanyl, furanylfentanyl, cyclopentylfentanyl, and ocfentanil. However, carfentanil is the most used recreationally. responsible for the worldwide opioid crisis, with special incidence in the United States [bib_ref] Novel Synthetic Opioids: An Opioid Epidemic Within an Opioid Epidemic, Lucyk [/bib_ref] [bib_ref] Increases in Drug and Opioid-Involved Overdose Deaths-United States, Rudd [/bib_ref]. In fact, from 2009 to 2019, a total of 77 NPS with opioid effects were reported by the UNODC. This phenomenon poses a challenge to public health authorities, as well as legislative authorities to avoid the dissemination, dependence, overdoses, and inappropriate use of these substances. Synthetic opioids are basically categorized into fentanyl and analogues, and non-fentanyl structured compounds.The former may be further divided into pharmaceutical fentanyls, which include fentanyl, for example, alfentanil and sufentanyl; and non-pharmaceutical fentanyls, which include fentanyl analogues and designer fentanyls, for instance ocfentanyl and bythyrfentanyl [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. Fentanyl, an analgesic analogue of phenopiperidine, is presented on [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. Fentanyl analogues [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref] followed fentanyl synthesis and were implemented in veterinary medicine, namely sufentanil, alfentanil, remifentanil, and carfentanil. Other analogues include acetylfentanyl, acryloylfentanyl, α-methylfentanyl, 3-methylfentanyl, furanylfentanyl, cyclopentylfentanyl, and ocfentanil. However, carfentanil is the most used recreationally. Substances such as AH-7921, U-47700, and analogues, MT-45, W-18, W-15, and IC-26 (analogue of methadone), are part of the non-pharmaceutical fentanyls family [bib_ref] Synthetic opioids, Beardsley [/bib_ref] [bib_ref] Maremmani, I. Non-Medical Use of Novel Synthetic Opioids: A New Challenge to..., Lovrecic [/bib_ref] [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. The selling of these new synthetic opioids occurs mostly online. Substances such as AH-7921, U-47700, and analogues, MT-45, W-18, W-15, and IC-26 (analogue of methadone), are part of the non-pharmaceutical fentanyls family [bib_ref] Synthetic opioids, Beardsley [/bib_ref] [bib_ref] Maremmani, I. Non-Medical Use of Novel Synthetic Opioids: A New Challenge to..., Lovrecic [/bib_ref] [fig_ref] Figure 1: Represents the chemical structure of some of piperazines [/fig_ref]. The selling of these new synthetic opioids occurs mostly online. It is not easy to estimate the prevalence of novel synthetic opioids, since the drug market is constantly changing [bib_ref] Maremmani, I. Non-Medical Use of Novel Synthetic Opioids: A New Challenge to..., Lovrecic [/bib_ref]. Oxycodone is a semi-synthetic opioid, used medically, but its recreational use and abuse has been reported. Commonly found in blotters, herbal smoking mixtures, fake medicine tablets or pills, these substances can be swallowed, inhaled (the vapors of heating the tablet), sniffed or even dissolved in water, and injected [bib_ref] Motives, diversion and routes of administration associated with nonmedical use of prescription..., Mccabe [/bib_ref]. Furthermore, fentanyl is also available in the form of patches. Nonetheless, the misuse of such patches can lead to toxicity; rectal insertion is an example [bib_ref] Rectal Insertion of Fentanyl Patches: A New Route of Toxicity, Coon [/bib_ref]. An increase in use of nasal sprays and e-liquids used in electronic cigarettes is also a concern to consider about these substances. From 2009-2020, over 65 new opioids were detected in Europe, and between 2012 and 2020, the vast majority were fentanyl derivatives. Moreover, in 2020, a report from EMCDDA included isotonitazene, a non-fentanyl synthetic opioid that can cause acute respiratory depression, leading to death. This substance was placed under international control in 2021. In fact, synthetic opioid abuse continues to increase while other substances decline. In 2020/2021, a total of 397 port-mortem cases were reported by the UNODC, and 14% fatalities were due to synthetic opioids. Moreover, 63% of reported cases involving these substances were due to the consumption of acetylfentanyl and 11% to carfentanil. Furthermore, nitazene opioids have recently been reported by the UNODC EWS. Undoubtedly, fentanyl precursors are emerging in the European market, with more than 33 kg of N-phenethyl4-piperidone (NPP) seized in Estonia. Other precursors, namely 4-piperidone monohydrate and 1-anilinopiperidine, were also seized in European countries. In 2018, around 70% of the reported opioids were fentanyl derivatives. The consumption of these compounds often leads to death, with few clinical admission scenarios. It is documented that these novel synthetic opioids have a higher potency than morphine [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. Moreover, opioids are known to wield their effects on opioid receptors, which mediate the actions of exogenous opioids. Similar to SCRA, the mechanism of action on these receptors is through G proteins, thus, mediating adenylyl cyclase by activation or inhibition [bib_ref] Opioid Crisis-An Emphasis on Fentanyl Analogs, Lutfy [/bib_ref]. Fentanyl and analogues are full agonists of μ-opioid receptors, estimating that fentanyl alone can have a potency of almost 50 to 100 times of that of morphine, while carfentanil has a potency 10,000 times higher than morphine and 100 times higher than fentanyl [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. After intravenous administration of fentanyl, respiratory depression can be reached within 2 to 5 min [bib_ref] Intravenous fentanyl kinetics, Mcclain [/bib_ref]. The elimination half time of this compound is 219 min. After about 7 to 13 min, maximal serum concentrations are reached, when this compound is administered intranasally, with effects lasting about 1 to 2 h [bib_ref] Intranasal fentanyl: From pharmacokinetics and bioavailability to current treatment applications, Panagiotou [/bib_ref] [bib_ref] Pharmacokinetics and Pharmacodynamics of Intranasal Versus Intravenous Fentanyl in Patients with Pain..., Foster [/bib_ref]. Biotransformation in the liver, by cytochrome P450 isozymes (CYP3A4), originates from It is not easy to estimate the prevalence of novel synthetic opioids, since the drug market is constantly changing [bib_ref] Maremmani, I. Non-Medical Use of Novel Synthetic Opioids: A New Challenge to..., Lovrecic [/bib_ref]. Oxycodone is a semi-synthetic opioid, used medically, but its recreational use and abuse has been reported. Commonly found in blotters, herbal smoking mixtures, fake medicine tablets or pills, these substances can be swallowed, inhaled (the vapors of heating the tablet), sniffed or even dissolved in water, and injected [bib_ref] Motives, diversion and routes of administration associated with nonmedical use of prescription..., Mccabe [/bib_ref]. Furthermore, fentanyl is also available in the form of patches. Nonetheless, the misuse of such patches can lead to toxicity; rectal insertion is an example [bib_ref] Rectal Insertion of Fentanyl Patches: A New Route of Toxicity, Coon [/bib_ref]. An increase in use of nasal sprays and e-liquids used in electronic cigarettes is also a concern to consider about these substances. From 2009-2020, over 65 new opioids were detected in Europe, and between 2012 and 2020, the vast majority were fentanyl derivatives. Moreover, in 2020, a report from EMCDDA included isotonitazene, a non-fentanyl synthetic opioid that can cause acute respiratory depression, leading to death. This substance was placed under international control in 2021. In fact, synthetic opioid abuse continues to increase while other substances decline. In 2020/2021, a total of 397 port-mortem cases were reported by the UNODC, and 14% fatalities were due to synthetic opioids. Moreover, 63% of reported cases involving these substances were due to the consumption of acetylfentanyl and 11% to carfentanil. Furthermore, nitazene opioids have recently been reported by the UNODC EWS. Undoubtedly, fentanyl precursors are emerging in the European market, with more than 33 kg of N-phenethyl4-piperidone (NPP) seized in Estonia. Other precursors, namely 4-piperidone monohydrate and 1-anilinopiperidine, were also seized in European countries. In 2018, around 70% of the reported opioids were fentanyl derivatives. The consumption of these compounds often leads to death, with few clinical admission scenarios. It is documented that these novel synthetic opioids have a higher potency than morphine [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. Moreover, opioids are known to wield their effects on opioid receptors, which mediate the actions of exogenous opioids. Similar to SCRA, the mechanism of action on these receptors is through G proteins, thus, mediating adenylyl cyclase by activation or inhibition [bib_ref] Opioid Crisis-An Emphasis on Fentanyl Analogs, Lutfy [/bib_ref]. Fentanyl and analogues are full agonists of µ-opioid receptors, estimating that fentanyl alone can have a potency of almost 50 to 100 times of that of morphine, while carfentanil has a potency 10,000 times higher than morphine and 100 times higher than fentanyl [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref]. After intravenous administration of fentanyl, respiratory depression can be reached within 2 to 5 min [bib_ref] Intravenous fentanyl kinetics, Mcclain [/bib_ref]. The elimination half time of this compound is 219 min. After about 7 to 13 min, maximal serum concentrations are reached, when this compound is administered intranasally, with effects lasting about 1 to 2 h [bib_ref] Intranasal fentanyl: From pharmacokinetics and bioavailability to current treatment applications, Panagiotou [/bib_ref] [bib_ref] Pharmacokinetics and Pharmacodynamics of Intranasal Versus Intravenous Fentanyl in Patients with Pain..., Foster [/bib_ref]. Biotransformation in the liver, by cytochrome P450 isozymes (CYP3A4), originates from norfentanyl [bib_ref] Fentanyl Metabolism by Human Hepatic and Intestinal Cytochrome P450 3A4: Implications for..., Labroo [/bib_ref] [bib_ref] Identification of Human Liver Cytochrome P-450 3A4 as the Enzyme Responsible for..., Tateishi [/bib_ref]. These isozymes are also involved in the metabolization of carfentanil (CYP3A5, CYP3A4, CYP2C8, and CYP2C9), forming different metabolites [bib_ref] Identification of human cytochrome P450 isozymes involved in the oxidative metabolism of..., Kong [/bib_ref]. In 2017, Watanabe et al. [bib_ref] In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic..., Watanabe [/bib_ref] predicted the structures of metabolites of acetylfentanyl, acrylfentanyl, furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl. The authors analyzed human urine samples to perform an in vivo analysis, and incubated the drugs with human hepatocytes for the in vitro analysis. The combination of these analyses allowed for the determination of 32 acetylfentanyl metabolites, a total of 14 acrylfentanyl metabolites and the same number of furanylfentanyl, and 17 4-fluoro-isobutyrylfentanyl metabolites. Metabolites of phase II were mostly observed for acetylfentanyl [bib_ref] In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic..., Watanabe [/bib_ref]. Recently, it was possible to identify in vitro ocfentanyl metabolites [bib_ref] Fatality involving ocfentanil documented by identification of metabolites, Allibe [/bib_ref]. A total of four metabolites were produced and results suggested that O-desmethylocfentanyl was the most abundant metabolite. A glucuronidated metabolite was also produced [bib_ref] Fatality involving ocfentanil documented by identification of metabolites, Allibe [/bib_ref]. AH-7921 can act as agonist of two opioid receptors (µ and κ) and possesses a high addictive potential [bib_ref] AH-7921: A new synthetic opioid of abuse, Coppola [/bib_ref]. There are not many studies about this substance; however, Swedish authorities have reported intoxications and deaths associated with the concomitant consumption of this substance and other psychoactive compounds [bib_ref] AH-7921: A new synthetic opioid of abuse, Coppola [/bib_ref]. In a review article about novel synthetic opioids, Prekupec et al. [bib_ref] Misuse of Novel Synthetic Opioids: A Deadly New Trend, Prekupec [/bib_ref] summarized that AH-7921 opioid can be consumed orally from low to high doses. Its effects last from 6 to 8 h, and after this period, effects can be observed up until 6 h [bib_ref] Misuse of Novel Synthetic Opioids: A Deadly New Trend, Prekupec [/bib_ref]. Furthermore, high doses are considered from 25 mg or higher. U-47700, the analogue of AH-7921, is considered to have a higher affinity to the µ-opioid receptor when compared to the other opioid receptors [bib_ref] Structure activity studies of two classes of beta-aminoamides: The search for kappa-selective..., Loew [/bib_ref]. Furthermore, the normal dose of this compound is around 7.5 to 15 mg, and it is approximately 7/8 times more potent than morphine [bib_ref] New synthetic opioids: Part of a new addiction landscape, Karila [/bib_ref] [bib_ref] Misuse of Novel Synthetic Opioids: A Deadly New Trend, Prekupec [/bib_ref] [bib_ref] Analysis of Novel Synthetic Opioids U-47700, U-50488 and Furanyl Fentanyl by LC-MS/MS..., Mohr [/bib_ref]. MT-45 is an agonist to δ and κ-opioid receptors. When consumed intravenously, it can be 11 times more lethal than morphine. A recent study aimed to obtain a pharmacological characterization of MT-45 in mice [bib_ref] In vitro and in vivo pharmacological characterization of the synthetic opioid MT-45, Bilel [/bib_ref]. Once again, studies in humans are lacking and they are fundamental to assess the risks of such compounds. Effects can be observed in the CNS, but cardiovascular and pulmonary effects have also been reported [bib_ref] Novel Synthetic Opioids: The Pathologist's Point of View, Frisoni [/bib_ref]. Sinicina et al. [bib_ref] Post-mortem review of fentanyl-related overdose deaths among identified drug users in Southern..., Sinicina [/bib_ref] have reviewed postmortem cases related to fentanyl. The authors reported that from 2005 to 2014, 242 overdoses in Bavaria, Germany, were related to fentanyl, and, in most cases, this substance was obtained via transdermal patches. The mean concentration in femoral blood was 16.9 µg/L [bib_ref] Post-mortem review of fentanyl-related overdose deaths among identified drug users in Southern..., Sinicina [/bib_ref]. On a Swedish project called STRIDA, it was possible to observe intoxications due to intake of different novel synthetic opioids (acrylfentanyl-mainly through nasal sprays, 4-fluoroisobutyrfentanyl and tetrahydrofuranfentanyl) [bib_ref] Intoxications involving acrylfentanyl and other novel designer fentanyls-results from the Swedish STRIDA..., Helander [/bib_ref]. The reported symptoms were anxiety, dizziness, nausea, vomiting, agitation, apnea, asystole, decreased consciousness, respiratory depression, and miosis. A death case due to brain edema is reported, involving the consumption of 4-fluoroisobutyrfentanyl. From the same project it was possible to evaluate intoxications related to butyrfentanyl, 4-fluorobutyrfentanyl, and fentanyl [bib_ref] Opioid intoxications involving butyrfentanyl, 4-fluorobutyrfentanyl, and fentanyl from the Swedish STRIDA project, Bäckberg [/bib_ref]. Recently, Wilde et al. [bib_ref] Acute severe intoxication with cyclopropylfentanyl, a novel synthetic opioid, Wilde [/bib_ref] , reported a severe intoxication case of cyclopropylfentanyl consumption; the symptoms reported after a 10 min intake were nausea, abundant sweating, and dyspnea, which culminated in miosis, coma, and respiratory insufficiency [bib_ref] Acute severe intoxication with cyclopropylfentanyl, a novel synthetic opioid, Wilde [/bib_ref]. Cyclopropylfentanyl was also found in another postmortem case. In this case, authors identified that the concentration of this compound increases in femoral blood for 18 h after the collection of the first sample, which suggests postmortem redistribution. Intravenous administration of acetyl fentanyl proved to be fatal on a case reported in 2015 by Cunningham et al. [bib_ref] Fatal Intoxication with Acetyl Fentanyl, Cunningham [/bib_ref]. The individual had a history of substance abuse; steroids were detected in urine, and acetyl fentanyl was detected at 235 ng/mL and 234 ng/mL in blood and urine, respectively. Moreover, pulmonary edema and cerebral edema were revealed in the autopsy. Pulmonary edema and lung injury were also observed on a death case associated with the consumption of ocfentanil [bib_ref] Ocfentanil overdose fatality in the recreational drug scene, Coopman [/bib_ref]. Guerrieri et al. conducted research on 40 fatal intoxications in which acrylfentanyl was a contributor or the main contributor to deaths. However, non-fatal cases are also mentioned in the literature. Hikin et al. reported numerous fatalities in the north of England, related to carfentanil, butytyl fentanyl, fluorobutyrylfentanyl, furanylfentanyl, alfentanil and fen-tanyl, being carfentanil, and fentanyl detected in seven cases. Carfentanil concentrations in blood ranged from 90 to 4004 pg/mL [bib_ref] Multiple fatalities in the North of England associated with synthetic fentanyl analogue..., Hikin [/bib_ref]. This same compound was also a target of a study in the United Kingdom and seven deadly cases were reported. The concentrations found in femoral postmortem blood ranged from 0.22 to 3.3 ng/mL [bib_ref] A Series of Deaths Involving Carfentanil in the UK and Associated Post-mortem..., Elliott [/bib_ref]. Over 6 months, Kronstrand et al. [bib_ref] Fatal Intoxications Associated with the Designer Opioid AH-7921, Kronstrand [/bib_ref] evaluated death cases related to the consumption of AH-7921. Research led to determination of a range of blood concentrations (0.03 to 0.99 µg/g). Although sometimes other compounds were also detected; this suggests that tolerance to this drug can vary, and deaths can occur with low and high doses. Moreover, possible metabolites have also been found [bib_ref] Fatal Intoxications Associated with the Designer Opioid AH-7921, Kronstrand [/bib_ref]. The first reported fatality case with U-47700 was described in 2016 by Elliot et al. [bib_ref] The first reported fatality associated with the synthetic opioid 3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) and..., Elliott [/bib_ref]. A concentration of 1.46 mg/mL was found in femoral blood, and the metabolites N-desmethyl-U-47700 and N,N-didesmethyl U-47700, and chemical structures were suggested [bib_ref] The first reported fatality associated with the synthetic opioid 3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) and..., Elliott [/bib_ref]. According to the EMCDDA, MT-45, can cause a wide range of effects, such as, somnolence, unconsciousness, high body temperature and vomiting, as well as tachycardia, decreased respiratory rate, hypoxia, hypo-and hypertension, seizures, low oxygen saturation, and miosis. Other symptoms such as hair depigmentation, hair loss, folliculitis, dermatitis, and dry eyes have also been reported [bib_ref] Acute skin and hair symptoms followed by severe, delayed eye complications in..., Helander [/bib_ref]. Overdose due to consumption of MT-45 was considered to be the cause of death in a case reported by Fels et al. [bib_ref] Two fatalities associated with synthetic opioids: AH-7921 and MT-45, Fels [/bib_ref] , and PB-22 and 5F-APINACA were also detected (although in very low amounts). The compound was determined in different matrices, and the concentrations varied between 24 and 1300 µg/mL, with the highest concentrations present in heart blood samples. These concentrations were much higher than those reported in the literature. In a study conducted by Papsun et al. [bib_ref] Analysis of MT-45, a Novel Synthetic Opioid, in Human Whole Blood by..., Papsun [/bib_ref] , it is reported that MT-45 was involved in one death, together with etizolam, with concentrations of 520 and 35 ng/mL, respectively. The death was attributed solely to the opioid since the concentration of etizolam was therapeutic. Tabarra et al. [bib_ref] Novel synthetic opioids-toxicological aspects and analysis, Tabarra [/bib_ref] recently published an interesting review about these compounds along with their toxicological aspects. ## Plant-based nps Plant-based NPS predominantly comprise alkaloids, which can induce a variety of effects, most commonly hallucinogenic, yet some can also induce relaxation [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Recent bionalytical methods for the determination of new psychoactive substances in biological..., Simão [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. Most of these NPS are often used for medicinal purposes or in rituals [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Overview of the major classes of new psychoactive substances, psychoactive effects, analytical..., Lukić [/bib_ref]. Only three plant-based NPS are under monitoring by the UNODC, namely Catha edulis, Salvia divinorum, and Mitragyna speciosa [bib_ref] Overview of the major classes of new psychoactive substances, psychoactive effects, analytical..., Lukić [/bib_ref]. Catha edulis (khat) is an indigenous plant from some Middle Eastern and West African countries, and among its contents approximately 40 alkaloids are present, mostly cathedulins (polyhydroxylated sesquiterpenes) and phenylalkylamines [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Overview of the major classes of new psychoactive substances, psychoactive effects, analytical..., Lukić [/bib_ref] [bib_ref] Chemistry, pharmacology, and toxicology of khat (catha edulis forsk): A review, Wabe [/bib_ref]. Three phenylalkylamines with high stimulant effect were detected: cathinone [S-(-)-cathinone], norephedrine [1R,2S-(-)-norephedrine] and cathine [1S,2S-(+)-norpseudoephedrine]. Khat is often consumed by chewing its fresh leaves, ingestion or smoking being relatively uncommon [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Chemistry, pharmacology, and toxicology of khat (catha edulis forsk): A review, Wabe [/bib_ref]. This is because cathinone is unstable during harvesting and drying processes. Between 2009 and 2012, this plant was among the most widely consumed NPS in EU member states, being the second most seized plant-based NPS worldwide in 2016. This plant was reported to be frequently misused by synthetic cathinone consumers given its lower risk [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. Although the main psychoactive compounds (cathinone and cathine) have been legislated and controlled since the 1971 UN Convention, several European countries, the United States, and Canada have started to introduce restrictions and/or legislation to control the entry of khat in their territories [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. The consumption of C. edulis has been associated with psychosis and aggressive behavior, as well as other symptoms, namely irregular blood pressure, tachycardia, urine retention, constipation, and insomnia, although many of these effects are still only described in animal studies [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Chemistry, pharmacology, and toxicology of khat (catha edulis forsk): A review, Wabe [/bib_ref]. Several medical conditions such as sleep disturbance, hypotension, and depression can be associated with withdrawal symptoms or a reduction in khat consumption, yet to date, it has not been possible to verify that consumption has been directly responsible for the death of any user [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. Salvia divinorum is another of the UNODC-controlled plant-based NPS, and originates from northeastern Mexico, and has been used for centuries for its hallucinogenic properties [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Pattern of use and subjective effects of Salvia divinorum among recreational users, González [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. This plant is consumed by chewing the leaves, or alternatively consumed as tea or even smoked, being easily purchased in smart shops [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Overview of the major classes of new psychoactive substances, psychoactive effects, analytical..., Lukić [/bib_ref] [bib_ref] Pattern of use and subjective effects of Salvia divinorum among recreational users, González [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] A unique natural selective kappa-opioid receptor agonist, salvinorin A, and its roles..., Cruz [/bib_ref] [bib_ref] Salvia divinorum: Toxicological aspects and analysis in human biological specimens, Margalho [/bib_ref]. In the United States, more than 1.8 million people have consumed this substance at least once in their lifetime, with reports from members of the United Nations in 2012 citing the plant as the third most consumed substance of its kind [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. Several European countries, the United States, and Canada have initiated legislation prohibiting consumption or controlling the entry into their territories [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Overview of the major classes of new psychoactive substances, psychoactive effects, analytical..., Lukić [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. The psychoactive effects of S. divinorum are promoted by the presence of salvinorin A, a dissociative hallucinogen with selective agonist effect for kappa opioid receptors (KOR), and whose mechanism of action is yet to be properly clarified [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Pattern of use and subjective effects of Salvia divinorum among recreational users, González [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. The effects promoted by salvinorin A are closely related to the route of administration. Psychoactive effects are observed within in a few seconds or minutes after inhalation or consumed through the buccal mucosa, even at low concentrations , which can last up to an hour [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Pattern of use and subjective effects of Salvia divinorum among recreational users, González [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. The half-life time of this compound is around 57 min, being longer in women [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. Short-term effects similar to the consumption of marijuana, LSD, or ketamine are common, including extracorporeal experiences, exacerbated relaxation, colorful visions, and loss of consciousness, yet to date, no deaths have been linked to consumption [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Pattern of use and subjective effects of Salvia divinorum among recreational users, González [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. Salvinorin A has been shown to possess some effects with therapeutic potential, for use in the treatment of drug addiction, pain, neurological and gastrointestinal diseases, and as anti-inflammatory agent [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] A unique natural selective kappa-opioid receptor agonist, salvinorin A, and its roles..., Cruz [/bib_ref] [bib_ref] Salvia divinorum: Toxicological aspects and analysis in human biological specimens, Margalho [/bib_ref]. There has been a growing concern over the recreational consumption of Mitragyna speciosa (kratom), a plant native to Southeast Asia [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Abuse liability and therapeutic potential of the Mitragyna speciosa (kratom) alkaloids mitragynine..., Hemby [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Mitragyna speciosa korth) for a new medicinal: A review of pharmacological and..., Firmansyah [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. It is commonly used for pain relief, to treat withdrawal symptoms in opiate users, hypertension, diarrhea, and coughing [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Abuse liability and therapeutic potential of the Mitragyna speciosa (kratom) alkaloids mitragynine..., Hemby [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Mitragyna speciosa korth) for a new medicinal: A review of pharmacological and..., Firmansyah [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. This plant is usually obtained fresh or dried, frequently consumed by chewing, drinking teas, or smoking. Several polyphenols, flavonoids, and glycosides have been identified among its constituents, as well as more than 40 alkaloids [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. The main alkaloid with psychoactive activity in kratom is mitragynine, followed by 7-hydroxymitragyne, mitraphylline, corynantheidine, speciogynine, paynantheine, and speciociliatine, which are also believed to be associated with the observed effects [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] Abuse liability and therapeutic potential of the Mitragyna speciosa (kratom) alkaloids mitragynine..., Hemby [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Mitragyna speciosa korth) for a new medicinal: A review of pharmacological and..., Firmansyah [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. This NPS is not yet listed in the schedule of the United Nations Convention on drugs. However, Mitragyna speciosa is controlled or classified as a narcotic by various entities in some countries in Southeast Asia, Oceania, the United States, and several European countries [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. There is considerable clinical interest in the plant's analgesic effect, since the affinity of mitragynine and 7-hydroxymitragynine for opioid receptors is well established [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. This plant has been used for several years in some regions of Asia to reduce fatigue and increase productivity at work, and also to treat coughing, pain, fever, diarrhea, hypertension, and diabetes. Kratom leaves have also been used as substitutes for opium, as well as in morphine withdrawal treatments [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref]. Mitragynine also interacts with δ and κ receptors, and a partial interaction of 7-hydroxymitragynine with κ receptors has also been reported [bib_ref] Psychoactive substances of natural origin: Toxicological aspects, therapeutic properties and analysis in..., Gonçalves [/bib_ref] [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref] [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref] [bib_ref] Mitragyna speciosa): Worldwide issues, Ramanathan [/bib_ref] [bib_ref] Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A..., Ya [/bib_ref]. The stimulant effects at low doses are thought to be associated with the same type of mechanism observed in low-dosed morphine [bib_ref] Here Today, Gone Tomorrow. and Back Again? A Review of Herbal Marijuana..., Rosenbaum [/bib_ref]. The set of effects begin approximately 5 to 10 min after consumption and may last for up to an hour [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. Several toxic effects are associated with recreational uses of Kratom, namely irritability, anxiety, and aggressiveness. When consumed for long periods, tremors, anorexia, psychosis, hyperpigmentation of the skin, and weight loss can be experienced [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. A number of fatalities have been associated with its consumption, with joint exposure to other substances being the most likely cause of death [bib_ref] Biomedical analysis of New Psychoactive Substances (NPS) of natural origin, Lo Faro [/bib_ref] [bib_ref] New psychoactive substances of natural origin: A brief review, Feng [/bib_ref]. # Conclusions The circumstances concerning drugs of abuse, and specifically NPS, are constantly changing. New substances capable of producing the effects desired by consumers appear daily, escaping legal control. Due to the relatively low cost and high potency of some of these substances, their use by marginalized groups, for example, people who are homeless or those with long-term and highly problematic drug use, is increasing. Moreover, since new psychoactive substances are more difficult to detect and identify in routine urine tests, they can be used by people who regularly undergo drug testing programs and want to avoid detection of their drug use. Examples of this are phencyclidine derivatives, opiates, and synthetic cannabinoids. Considering that each country has its own legislation on these new substances, different mechanisms exist concerning improving public health or social policies. There is no doubt that EWS plays a central role in supporting readiness and coordinated responses to the NPS problem, both at the national and international level. Awareness campaigns aimed at the younger population highlighting the deleterious effects of these substances (e.g., schools, social media, and media) may also have an important effect. The imprisoned population could also be targeted with such actions. Carrying out studies that identify the mechanism for action of these substances, and their effects, and consequences in the short and long term is undoubtedly a valuable tool in the toxicological field. In addition, laboratorial developments allowing the rapid identification of these substances, mainly at the emergency room level, would help. Furthermore, multidisciplinary research in the areas of epidemiology and prevention, and public health allow the development of adequate measures to monitor consumption and implement public health education policies. All these factors will certainly contribute to the implementation of social and public health measures to efficiently face the scourge of NPS consumption worldwide. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19084869/s1, Table S1: Classification of piperazines; : Intoxications with piperazines; : Examples of natural and synthetic tryptamines; : Classes of SCRA and their chemical characterization; : Phencyclidine-type substances; : Case report intoxications with phencyclidine-type substances. References [bib_ref] Toxic effects of BZP-based herbal party pills in humans: A prospective study..., Gee [/bib_ref] [bib_ref] Nephrotoxicity of BZP-based herbal party pills: A New Zealand case report, Alansari [/bib_ref] [bib_ref] Multiorgan failure from 1-benzylpiperazine ingestion legal high or lethal high, Gee [/bib_ref] [bib_ref] Acute psychosis following ingestion of 'Rapture', Austin [/bib_ref] [bib_ref] Tödliches hirnödem nach einnahme von ecstasy und benzylpiperazin, Balmelli [/bib_ref] [bib_ref] Acute chlorophenylpiperazine overdose: A case report and review of the literature, Kovaleva [/bib_ref] [bib_ref] Toxicity from the recreational use of 1-benzylpiperazine, Gee [/bib_ref] [bib_ref] Methoxetamine associated reversible cerebellar toxicity: Three cases with analytical confirmation, Shields [/bib_ref] are cited in the supplementary materials. [fig] Figure 1: Represents the chemical structure of some of piperazines.The two main structural groups that form the piperazine-based compounds are 1benzylpiperazine and 1-phenylpiperazine (Supplementary Material Table S1 andFigure 1)[10,13,31,32,[35][36][37][38][39][40]. [/fig] [fig] Figure 2: Chemical structure of rasagiline. [/fig] [fig] Figure 3: Chemical structure of amphetamine (left) and 2-AI (right). [/fig] [fig] Figure 4: Chemical structures of synthetic aminoindanes. [/fig] [fig] Figure 5: Chemical structure of amphetamine and cathinone. [/fig] [fig] Figure 6: Chemical structures of methamphetamine, MDMA, ephedrone, and methylone. [/fig] [fig] Figure 7: General chemical structure of synthetic cathinones. [/fig] [fig] Figure 8: Some of the most common synthetic cathinones. [/fig] [fig] Figure 9: Chemical structure of phenylethylamine. [/fig] [fig] Author: Contributions: Conceptualization, E.G., M.B., M.A. (Maristela Andraus), and L.A.P.; methodology, A.Y.S., M.A. (Mónica Antunes), E.C., E.A., L.M.R., P.O., A.T.B., H.M., and T.R.; writing-original draft preparation, A.Y.S., M.A. (Mónica Antunes), E.C., E.A., L.M.R., P.O., A.T.B., H.M., T.R., and E.G.; writing-review and editing, A.Y.S., M.A. (Mónica Antunes), E.G., M.B., T.R., M.A. (Maristela Andraus), and L.A.P.; supervision, E.G., M.B., and M.A. (Maristela Andraus). All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology (FCT)/MCTES. It was also supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy-i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Ana Y. Simão and Mónica Antunes acknowledge the PhD fellowships from FCT (2020.09070.BD and 2020.05765.BD, respectively). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing is not applicable to this article. [/fig]

Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices. # Introduction Developing surface finishes to encourage osteoblastogenesis is a continuing theme of contemporary bone biomaterials research. Enhancing human osteoblast (hOB) formation and maturation at prosthetic surfaces is predicted to secure superior implant integration and longevity. Novel ways of fabricating unique substrates includes coating bone biomaterials with small biological agents. Importantly the selected molecules should target hOBs and their bone marrow-derived stromal cell (BMSC) precursors. Particularly attractive are agents that participate in signalling co-operation of "cross-coupling" with key molecules central to bone formation, health and homeostasis. Candidate factors fulfilling these criteria are the simple bioactive lysophosphatidic acids (LPAs) and/or their more stable, phosphatase-resistant analogues. The term LPA (1-acyl-2-hydroxy-sn-glycero-3-phosphate) actually refers to a group of lysophospholipids that are characterized as having a fatty acyl chain, glycerol backbone and a phosphate head group, different LPAs vary according to chain length and degree of saturation. The different LPAs interact with a wide range of G protein-coupled receptors (GCPR), and members of the LPA family are involved in diverse physiological activities such as wound healing, cell survival, apoptosis, motility and differentiation (reviewed in [bib_ref] LPA receptors: subtypes and biological actions, Choi [/bib_ref]. Skeletal cells, including hOBs and BMSCs, are also targets for LPAs [bib_ref] The emerging role of lysophosphatidic acid (LPA) in skeletal biology, Blackburn [/bib_ref]. Of particular relevance to bone formation is the discovery that LPA co-operates with the active metabolite of vitamin D 3 , 1,25-dihydroxy vitamin D [bib_ref] Lysophosphatidic acid cooperates with 1alpha,25(OH)2D3 in stimulating human MG63 osteoblast maturation, Gidley [/bib_ref] , to secure hOB maturation [bib_ref] Lysophosphatidic acid cooperates with 1alpha,25(OH)2D3 in stimulating human MG63 osteoblast maturation, Gidley [/bib_ref] [bib_ref] Lysophosphatidic acid, human osteoblast formation, maturation and the role of 1α,25-Dihydroxyvitamin D3..., Mansell [/bib_ref] [bib_ref] 25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure..., Lancaster [/bib_ref]. It is the mature osteoblast phenotype that is responsible for the provision of a mechanically robust, mineralised matrix. The signalling cross-coupling that occurs between LPA and active vitamin D 3 metabolites culminates in synergistic increases in alkaline phosphatase (ALP), an enzyme absolutely essential for bone matrix calcification [bib_ref] Physiological role of alkaline phosphatase explored in hypophosphatasia, Whyte [/bib_ref]. These features of LPA, its small size and ability to co-operate with 1,25D make it an especially desirable molecule for the functionalisation of titanium and hydroxyapatite, two widely used bone biomaterials. The only other molecules reported to cooperate with 1,25D in stimulating hOB maturation are TGF-β [bib_ref] Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation..., Bonewald [/bib_ref] and epidermal growth factor [bib_ref] Epidermal growth factor and calcitriol synergistically induce osteoblast maturation, Yarram [/bib_ref]. Their larger size and greater cost make them less desirable contenders for biomaterial conjugation and they are less likely to withstand conventional sterilisation protocols. In addition to its reported effects on hOBs, 16:0 MPPA, an LPA with an unsaturated C16 chain, has been reported to inhibit virulence factor production and biofilm formation of the human opportunistic pathogen Pseudomonas aeruginosa [bib_ref] Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin,..., Laux [/bib_ref]. [bib_ref] Lipid phosphate phosphatases and signaling, Brindley [/bib_ref] :0 MPPA has together with several other phospholipids such as dipalmitoyl phosphatidyl serine, dipalmitoyl phosphatidic acid and monomyristoyl phosphatidic acid been shown to sensitize otherwise resistant P. aeruginosa isolates to the actions of betalactams [bib_ref] Specific phospholipids enhance the activity of beta-lactam antibiotics against Pseudomonas aeruginosa, Krogfelt [/bib_ref]. We have also shown that 16:0 MPPA and monopalmitoyl phosphatidyl choline are antimicrobial against a range of gram positive bacteria such as Staphylococcus aureus and Enterococcus spp. Collectively LPA-functionalised devices could be beneficial in two important ways; the enhancement of early osseointegration by promoting hOB maturation at the surface and secondly, a substrate that is less appealing to bacteria at the time of implantation. Biological coating of contemporary bone biomaterials can be particularly challenging, often requiring time consuming, complex and costly procedures. However there is an attractive, facile method for functionalising titanium devices; the use of alkane phosphonic acids (APAs) which have a natural, high affinity for metal oxides [bib_ref] Surface modification using phosphonic acids and esters, Queffélec [/bib_ref] including titania (TiO 2 ), the natural coating of titanium. The bonds formed between TiO 2 and APAs are robust, iono-covalent in nature and are superior to those formed using silanes as no further cross-linking is required. The nature of the APA-TiO 2 bond has been reported to be mono and/or bidentate and can also include electrostatic interactions [bib_ref] Theoretical Characterization of the Indium Tin Oxide Surface and of Its Binding..., Paramonov [/bib_ref] [bib_ref] Self-assembled monolayers and titanium dioxide: From surface patterning to potential applications, Paz [/bib_ref]. Herein we report the facile functionalisation of Ti using octadecylphosphonic acid (ODPA) for the subsequent attachment of 16:0 MPPA and a palmitoyl, phosphatase-resistant LPA analogue, O-2-(hexadecyloxy)-3-methoxypropyl O-hydrogenphosphorothioate (16:0 OMPT) [bib_ref] Enantioselective responses to a phosphorothioate analogue of lysophosphatidic acid with LPA3 receptor-selective..., Qian [/bib_ref]. The rationale for using a phosphatase-resistant LPA species over 16:0 MPPA is to produce a Ti surface finish with increased "residence time" of the tethered bioactive as native LPAs are susceptible to rapid hydrolysis by lipid phosphate phosphatases [bib_ref] Lipid phosphate phosphatases and signaling, Brindley [/bib_ref]. These modified surfaces were evaluated for their ability to support 1,25D-induced hOB maturation. In parallel the same surfaces were exposed to S. aureus to investigate the effect of these functionalised materials on bacterial attachment and growth. ## Materials and methods general Unless stated otherwise, all reagents were of analytical grade from Sigma-Aldrich (Poole, UK). Stocks of 16:0 MPPA (Cambridge Bioscience, Cambridge, UK) were prepared in 1:1 ethanol: tissue culture grade water to a final concentration of 10 mM and stored at -20°C. Likewise, 1,25D (100 micromolar) was prepared in ethanol and stored at -20°C. Stable, phosphataseresistant 16:0 OMPT was synthesized at The University of Utah as detailed below. The compound was reconstituted in 1:1 ethanol:tissue culture grade water to a final concentration of 10 mM and stored at -20°C. Octadecylphosphonic acid (ODPA) was dissolved in anhydrous tetrahydrofuran to a final concentration of 1 mM in glass and stored at ambient temperature. Synthesis of O-2-(hexadecyloxy)-3-methoxypropyl Ohydrogenphosphorothioate (16:0 OMPT) 2-(hexadecyloxy)-3-methoxypropan-1-ol. A solution of 1-trityl-3-methyl-glycerol (2.537 g, 7.29 mmole) in 40 ml of dimethylformamide (DMF) was added sodium hydride (60% in mineral oil, 335 mg, and 1.15 eq. 8.39 mmole). Then the reaction mixture was stirred at room temperature for 1 hour before 1-bromohexadecane (2.67 g, 8.75 mmole, 1.2 eq.) in 10 mL DMF and tributylammonia iodide (0.1 eq, 0.73 mmole, 269 mg) were added. The mixture was stirred overnight at room temperature. Then 1N HCl (60 mL) was added to quench the reaction, and the mixture was extracted with 100 mL EtOAc twice. The organic extract was washed with saturated NaHCO 3 solution, followed by a saturated NaCl solution. Solvent was removed at reduced pressure and the crude mixture was purified on silica column (EtOAc:hexane 5/95) to give crude hexadecyl ether (1.537 g, 2.67 mmole, 37%), which was directly dissolved into 30 mL dichloromethane and then 3 mL trifluoroacetic acid was added. The reaction mixture was stirred for 1 hour at room temperature. Then solid sodium bicarbonate was added slowly to neutralize the reaction mixture, followed by 100 mL of water. The organic layer was separated and the water layer was extracted by dichloromethane (100 mL) twice. All organic layers were combined and dried over sodium sulphate. The organic solvent was concentrated and the residue was purified on silica column (EtOAc:hexane 5/95, 15/85) to give 2-(hexadecyloxy)-3-methoxypropan-1-ol (722 mg, 2.19 mmole, 82%) as a waxy solid. [bib_ref] LPA receptors: subtypes and biological actions, Choi [/bib_ref] After 48 hours, the reaction mixture was concentrated and the residue was purified on silica column (EtOAc:CH 3 OH, 9:1) to afford 91 mg of the ammonium salt of the 16:0 analogue of OMPT as a light yellow oil (0.21 mmole, 78%). The monosodium salt was obtained via ion exchange resin (Dowex 200 Na + form). [bib_ref] LPA receptors: subtypes and biological actions, Choi [/bib_ref] ## Lysophosphatidate-functionalisation of ti specimens Titanium (Ti) discs (12.7 mm diameter and 2.5 mm thickness) of orthopaedic grade titanium were a generous gift from Corin (Cirencester, UK). To encourage oxide formation Ti discs were either oven baked at 160°C overnight (~18 hours) or treated with Piranha solution as described by us previously [bib_ref] Lysophosphatidic acid-functionalised titanium as a superior surface for supporting human osteoblast (MG63)..., Mansell [/bib_ref]. Briefly, Piranha solution was prepared by combining equal volumes (20 mL) of ice-cold concentrated sulphuric acid and 30% (w/v) hydrogen peroxide. [Please note that Piranha solution is highly corrosive and oxidative and should be handled with care]. Once mixed the resultant solution was allowed to reach room temperature before use. Ti discs were steeped in Piranha solution for 2 hours under gentle stirring at ambient temperature. Discs were subsequently washed and patted dry. Oven-baked Ti, Piranha-treated Ti and unpre-treated Ti discs were then immersed in 1 mM ODPA in tetrahydrofuran (THF) for 1 hour at ambient temperature. The Ti specimens were allowed to dry in a fume cupboard at ambient temperature, and then given two 5-minute rinses in THF, allowed to dry followed by baking overnight at 160°C. Each Ti disc was then placed into a well of a 24-well tissue culture plate and bathed in 1 ml of 100 micromolar 16:0 OMPT in 50% aqueous (tissue culture grade water) ethanol, and samples left overnight at ambient temperature. Then the discs were rinsed in tissue culture-grade water and allowed to dry, at ambient temperature in a tissue-culture cabinet. ## Human mg63 osteoblasts Human osteoblast-like cells (MG63, ECACC, item code: 86051601) were cultured in conventional tissue culture flasks (250 mL, Greiner, Frickenhausen, Germany) in a humidified atmosphere at 37°C and 5% CO 2 . Although osteosarcoma derived, MG63 cells exhibit features in common with human osteoblast precursors or poorly differentiated osteoblasts. Specifically, these cells produce type I collagen with no or low basal osteocalcin (OC) and ALP [bib_ref] Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of..., Clover [/bib_ref]. However, when MG63 cells are treated with 1,25D, cell proliferation is inhibited and there is an accompanying, enhanced expression of OC. When MG63 cells are co-stimulated with 1,25D and selected growth factors, e.g., LPAs, then there is a marked, synergistic increase in ALP reflecting transition to the mature phenotype. Thus, the application of these cells to assess the compatibility of LPA-functionalised biomaterials for orthopaedic applications is entirely appropriate. MG63 cells were grown to confluence in Dulbecco's modified Eagle medium (DMEM)/F12 nutrient mix (Gibco, Paisley, Scotland) supplemented with sodium pyruvate (1 mM final concentration), L-glutamine (4 mM), streptomycin (100 ng/mL), penicillin (0.1 units/mL) and 10% v/v foetal calf serum (Gibco, Paisley, Scotland). The growth media was also supplemented with 1% of a 100x stock of non-essential amino acids. Once confluent, MG63 cells were subsequently dispensed into 24-well plates (Greiner, Frickenhausen, Germany) containing control and 16:0 OMPT functionalised Ti surfaces. In each case, wells were seeded with 1 mL of a 4x10 4 cells/mL suspension (as assessed by haemocytometry) in serum free medium supplemented with 100 nM 1,25D. Cells were then cultured for 3 days, the Ti specimens removed and transferred to clean plates to ascertain cell number and total ALP activity for cells associated with the metal samples. ## Enumeration of mg63 cells An assessment of cell number was performed using a combination of the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt (MTS, Promega, UK) and the electron-coupling reagent phenazine methosulphate (PMS). Each compound was prepared separately in pre-warmed (37°C) phenol red-free DMEM/F12, allowed to dissolve, and then combined so that 1 mL of a 1 mg/mL solution of PMS was combined to 19 mL of a 2 mg/mL solution of MTS. A stock suspension of MG63 cells (1x10 6 cells/mL) was serially diluted in growth medium to give a series of known cell concentrations down to 2.5x10 4 cells/mL. Each sample (0.5 mL in a microcentrifuge tube) was spiked with 0.1 mL of the MTS/PMS reagent mixture and left for 45 minutes within a tissue culture incubator. Once incubated, the samples were centrifuged at 900 rpm to pellet the cells and 0.1 mL of the supernatants dispensed onto a 96-well microtitre plate and the absorbance read at 492 nm using a multiplate reader. Plotting the absorbances against known cell number, as assessed initially using haemocytometry, enabled extrapolation of cell numbers for the experiments described herein. ## Total alkaline phosphatase activity An assessment of ALP activity is reliably measured by the generation of p-nitrophenol (p-NP) from p-nitrophenylphosphate (p-NPP) under alkaline conditions. The treatment of cells to quantify ALP activity was similar to that described by us recently [bib_ref] The role of lysophosphatidic acid on human osteoblast formation, maturation and the..., Lancaster [/bib_ref]. Briefly, the MTS/PMS reagent was removed and the monolayers incubated for a further 15 minutes in fresh phenol red-free DMEM/F12 to remove the residual formazan. Following this incubation period, the medium was removed and the monolayers lysed with 0.1 mL of 25 mM sodium carbonate (pH 10.3), 0.1% (v/v) Triton X-100. After 2 minutes, each well was treated with 0.2 mL of 15 mM p-NPP (di-Tris salt, Sigma, UK) in 250 mM sodium carbonate (pH 10.3), 1 mM MgCl 2 . Lysates were then left under conventional cell culturing conditions for 1 h. After the incubation period, 0.1 mL aliquots were transferred to 96-well microtitre plates and the absorbance read at 405 nm. An ascending series of p-NP (25-500 micromolar) prepared in the incubation buffer enabled quantification of product formation. Unless stated otherwise, total ALP activity is expressed as the mean micromolar concentration of p-NP per 100,000 cells, as extrapolated from the MTS/PMS assay described above. ## Growth kinetics of s. aureus Unless stated otherwise all media components used for growth of bacteria were from Sigma. Bacterial growth of the Methicillin Resistant strain S. aureus 43484 [bib_ref] Efficacy of topical and systemic antibiotic treatment of meticillin-resistant Staphylococcus aureus in..., Lundberg [/bib_ref] was investigated in a 96 well microtiter plate. A single colony of strain MRSA 43484 from an overnight LB plate was inoculated into Luria Broth (LB) media and grown until exponential phase (around 4 hours) at 37°C and shaking of 200 rpm. Bacteria were then diluted 100 times in LB or minimal media as outlined and added calcium chloride when described. Minimal media consisted of M9 minimal media containing 2% glycerol, 0.5% cas-amino acids, 0.2 microgram/mL niacin, and 0.2 microgram/mL thiamine, and 2 mM magnesium chloride. EDTA, 16:0 OMPT and 16:0 MPPA were dissolved and diluted in PBS in two times the final concentration in a 96 well plate. Then the diluted compounds were mixed with equal amounts of the bacterial culture in the relevant media (LB or minimal media). Absorbance at OD 450 nm of the 96 well microtiter plate was read every 20 minutes the following 15 hours using a BMG Labtech FLUOstar OPTIMA Microplate Reader running at 37°C. ## Flow cytometry A single colony of MRSA 43484 was inoculated into LB. After 4 hours incubation, this culture was used to inoculate LB media containing either 16:0 OMPT, 16:0 MPPA or EDTA at the concentrations specified or control, and incubated over night at 37°C, shaking at 200 rpm. Bacterial cultures were then washed in PBS (GIBCO, Grand Island, NY) and adjusted to approximately OD 600 nm 0,002. The bacterial suspension were then stained with 500nM propidium iodide for five minutes at 30°C before analysis on an Accuri C6 standard set-up flow cytometer (Accuri, Ann Arbor, MI). Initial gating of bacteria was performed using forward scatter (FSC-H) and side scatter (SSC-H). The gated population was further analysed using excitation of PI at 488 nm and emission at 670/LP nm (FL-3). The flow cytometer was operated at the Slow Flow Rate setting (14 microliter sample/minute). ## Bacterial adhesion to functionalised ti A single colony of MRSA 43484 was inoculated into LB media and grown overnight to stationary phase at 37°C and shaking (200 rpm). The culture was then washed once in LB and diluted to OD 600 nm 0,010 (corresponding to approximately 3x10 7 CFU pr. mL) with fresh LB. 1 mL pr. well of the bacterial suspension was added to the wells of a 24 well plate (Nunc, Roskilde, Denmark) and Ti discs were added to the wells. The 24 well plate with Ti discs and bacteria was incubated for 8 h at 37°C. The Ti discs were then washed briefly in PBS and transferred to new wells with sterile PBS to loosen adhering bacteria. The plate was then stored at 4°C for 24-72 hours. The Ti discs were removed and CFU pr. mL of the detached bacteria (in PBS) was determined by spot plating (in duplicate). Four discs per group (control, OMPT or MPPA) were used and the experiment performed four times. To determine whether the coating on the Ti discs remained their activity after one time use, in one experiment the discs were reused: after the experiment the slides rinsed with soap and distilled water, padded dry and finally autoclaved and used as described above. During the rinsing procedure, mixing of the Ti discs was avoided by using separate glass vials for the different types of surface modification. ## Scanning electron microscopy Bacterial adhesion was performed as described above, but instead of detaching bacteria by incubation in PBS, the briefly rinsed Ti discs with adhering bacteria were fixed in 2% glutaraldehyde in PBS, and postfixed in 1% OsO4. Samples were then dehydrated in ethanol, critical point-dried using CO 2 , and sputter-coated with gold. Samples were investigated using a Philips XL Feg30 scanning electron microscope operated at 2 kV. The number of adherent bacteria pr. micrometer 2 was determined by manual counting of resulting images. Three images captured from different, random locations at the Ti disc were counted for each condition. For each sample the number of bacteria adhering to at least 10000 micrometer 2 was determined. The SEM experiment was performed once. ## Statistical analyses Unless stated otherwise, all the cell culture experiments described above were performed three times and all data were subject to a one-way analysis of variance (ANOVA) to test for statistical significance as we have reported previously [bib_ref] 25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure..., Lancaster [/bib_ref]. When a p value of < 0.05 was found, a Tukey multiple comparisons post-test was performed between all groups. All data are expressed as the mean together with the standard deviation. Investigations of adhesion of MRSA to Ti discs were also performed at least three times unless otherwise stated and data were analysed with a one-way ANOVA using Dunnett's multiple comparison post-test between control and treatment groups (functionalised Ti discs) for p values < 0.05. # Results ## 16:0 ompt and 1,25d co-operate to promote human osteoblast maturation Cell number increased significantly in the presence of 16:0 OMPT (10 micromolar), while 1,25D (100 nM) caused a significant reduction in cell number. However, the combined effect of 16:0 OMPT and 1,25D gave cell numbers similar to untreated cells [fig_ref] Fig 2: Growth and maturation of human [/fig_ref]. After three days of culture, the extent of maturation of the osteoblasts in response to the active vitamin D 3 metabolite; 1,25D and 16:0 OMPT was determined by measurement of alkaline phosphatase activity via quantification of p-NP from p-NPP. Co-stimulation of MG63 osteoblasts with 1,25D and 16:0 OMPT led to clear, synergistic, increases in ALP activity, as supported by elevated p-NP production [fig_ref] Fig 2: Growth and maturation of human [/fig_ref]. ## 16:0 ompt-functionalised ti enhances 1,25d-induced mg63 maturation Human MG63 osteoblasts were seeded onto ODPA or ODPA-16:0 OMPT modified Ti specimens. Ti specimens subjected to ODPA-16:0 OMPT functionalisation caused a significantly higher degree of 1,25D-induced osteoblast maturation than ODPA alone [fig_ref] Fig 3 16: [/fig_ref]. Moreover, pretreatment of the Ti specimens before surface modification either by baking or Piranha treatment gave even higher levels of p-NP ( ÃÃ p<0.001 versus ODPA control Ti) and thus MG63 osteoblast maturation than direct surface modification (16:0 OMPT ambient, Ã p<0.01 versus ODPA control Ti). No difference was found between baking and Piranha pretreatment with regard to MG63 maturation. Baking as pre-treatment option was used in all subsequent modifications for the microbiological studies described in the following. Antibacterial effects of 16:0 MPPA, 16:0 OMPT and EDTA We have previously shown that 16:0 MPPA is antibacterial against a range of bacteria. In order to investigate whether the artificial LPA analogue 16:0 OMPT had similar properties we were equally divided as follows: some were subjected to either Piranha solution (OMPT Piranha) or baked at 160°C overnight (OMPT baked) prior to steeping in ODPA for an hour under ambient conditions. Recovered samples were then exposed to 16:0 OMPT (0.1mM in 50% aqueous ethanol) overnight. Some discs were treated with ODPA alone (control) and others ODPA followed by 16:0 OMPT with no prior baking or Piranha treatment (OMPT ambient). Each group of discs were transferred to multiwell tissue culture plates and seeded with MG63 osteoblasts in a serum-free culture medium supplemented with 100nM 1,25D. After three days of culture an assessment of total ALP activity was performed using p-nitrophenylphosphate as substrate and quantification of p-nitrophenol (p-NP). The concentration of p-NP for cells upon ODPA/16:0 OMPT-functionalised Ti is greater than for cells on control Ti (*p<0.01 compared to control Ti). The extent of maturation, as supported by raised p-NP, is significantly greater still (**p<0.001 compared to control Ti) by prior baking/Piranha treatment of the Ti samples. The data presented are a representative of three independent experiments. In each case the data shown are the mean micromolar concentration of p-NP normalised to 10 5 cells + SD of 4 replicate samples. ## Flow cytometry To investigate whether 16:0 OMPT and 16:0 MPPA were bactericidal or bacteriostatic we investigated the uptake of the nonviable dye propidium iodide (PI) by MRSA grown in the presence of these compounds. The concentration of MPPA, OMPT and EDTA were adjusted to achieve limited growth; that is OD 600 1.5 compared to untreated bacteria that achieved an OD 600 of 3.0. Functionalisation using a linker such as ODPA should make the binding of compounds to the surface very durable through interaction of the hydrophobic alkyl chains of both the ODPA and the LPA/LPA analogue. To test this, we reused the Ti discs in a new experiment after careful rinsing and autoclaving of the Ti discs. [fig_ref] Fig 8: Lysophosphatidiate-functionalised titanium inhibits MRSA adhesion [/fig_ref] that in the reuse experiment the antiadhesive effect reached the same level as the first round of the experiment. As mentioned above we found that adding Ca 2+ abolished the growth inhibitory effect of 16:0 MPPA, and we wanted to investigate whether extra Ca 2+ would also affect the anti-adhesive effect of 16:0 [fig_ref] Fig 8: Lysophosphatidiate-functionalised titanium inhibits MRSA adhesion [/fig_ref] shows that growth in the media above the functionalised Ti discs [fig_ref] Fig 8: Lysophosphatidiate-functionalised titanium inhibits MRSA adhesion [/fig_ref] were not affected, suggesting that low/no 16:0 MPPA or 16:0 OMPT was released from the Ti discs into the media. ## Scanning electron microscopy Scanning Electron Microscopy (SEM) was performed on functionalised Ti discs with adherent MRSA bacteria. Examples of resulting images are shown in [fig_ref] Fig 9: Lysophosphatidate-functionalisation of titanium deters bacterial attachment and alters bacterial surface morphology [/fig_ref] Manual counting of adherent bacteria/micrometerm 2 showed that significantly fewer bacteria adhered to the 16:0 OMPT and 16:0 MPPA functionalised Ti compared to the control. Interestingly, the images showed that while the bacteria adhering to the control Ti discs had a very rough appearance, bacteria adhering the 16:0 OMPT and especially the 16:0 MPPA functionalised Ti discs appeared smooth and with less extracellular matrix material. # Discussion Developing novel biomaterial coatings that enhance early osseointegration yet deter the attachment of bacteria are especially desirable properties for future orthopaedic and dental implant devices. The simple pleiotropic lipid, LPA and/or selected LPA analogues, could represent candidate molecules for coating Ti. Over a decade ago Laux and colleagues [bib_ref] Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin,..., Laux [/bib_ref] described how 16:0 MPPA (palmitoyl LPA) inhibited the growth and virulence of P. aeruginosa. 16:0 MPPA is a naturally occurring LPA found in biological fluids including bronchoalveolar lavage (BAL), serum and plasma [bib_ref] A novel highly potent autotaxin/ ENPP2 inhibitor produces prolonged decreases in plasma..., Saga [/bib_ref] [bib_ref] Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis, Tokumura [/bib_ref] [bib_ref] Autotaxin production of lysophosphatidic acid mediates allergic asthmatic inflammation, Park [/bib_ref]. Interestingly, exposure of bacteria to this lipid made them increasingly susceptible to certain antibiotics. This particular lipid species sits amongst several structurally similar lysophospholipids that serve as signalling molecules to most mammalian cell types [bib_ref] The ins and outs of lysophosphatidic acid signaling, Moolenaar [/bib_ref] In the context of human osteoblast biology LPAs co-operate with 1,25D to enhance their maturation [bib_ref] Lysophosphatidic acid cooperates with 1alpha,25(OH)2D3 in stimulating human MG63 osteoblast maturation, Gidley [/bib_ref] [bib_ref] Lysophosphatidic acid, human osteoblast formation, maturation and the role of 1α,25-Dihydroxyvitamin D3..., Mansell [/bib_ref] an event synonymous with bone tissue formation. Given the reported effects of 16:0 MPPA on bacteria suggest LPA/LPA analogues as potential adjuncts in a bone regenerative setting and one way in which this could be realised is by coating biomaterials with LPA/LPA analogues. Such a "dual-action" coating would be particularly appealing to reduce potential infection risk of implantable devices whilst encouraging superior early osseointegration. Human osteoblasts and bone marrow stromal cells express at least three different LPA receptor types. When stimulated with LPA/LPA analogues effects ranging from proliferation, fibronectin binding, cytoskeletal reorganization and differentiation have been reported [bib_ref] The emerging role of lysophosphatidic acid (LPA) in skeletal biology, Blackburn [/bib_ref]. In this particular investigation we found that 16:0 OMPT stimulated MG63 cell growth. When the same cells were co-stimulated with 1,25D and 16:0 OMPT cell growth was modestly attenuated and there was a synergistic increase in total ALP activity indicating a change towards a more mature or differentiated phenotype. All of these changes essentially mirror the effects we have seen for other LPA species on this cell type. In this study we also show that the functionalisation of Ti with 16:0 OMPT, enhances 1,25D-induced MG63 maturation, as supported by the greater total ALP activity. The effect of 16:0 OMPT functionalisation on maturation of MG63 is particularly encouraging; mature osteoblasts are responsible for bone matrix synthesis and mineralisation and if these events can be enhanced at the Ti surface then this could facilitate the process of early osseointegration. We have previously shown that 16:0 MPPA sensitises otherwise betalactam resistant P. aeruginosa to the actions of ampicillin [bib_ref] Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin,..., Laux [/bib_ref] [bib_ref] Specific phospholipids enhance the activity of beta-lactam antibiotics against Pseudomonas aeruginosa, Krogfelt [/bib_ref]. We have also shown that this LPA is antibacterial against a number of Gram positive bacteria including S. aureus. Here we further show that the phosphatase resistant LPA analogue, 16:0 OMPT, is antibacterial against MRSA strain 43484. The mechanism by which the two compounds exert their effects is unclear. We have previously reported that 16:0 MPPA precipitates in the presence of the divalent cations calcium and magnesium and that precipitation is most noticeable with the former ion. These observations imply that 16:0 MPPA can function as a chelator [bib_ref] Specific phospholipids enhance the activity of beta-lactam antibiotics against Pseudomonas aeruginosa, Krogfelt [/bib_ref]. Due to the clear structural similarities between 16:0 MPPA and 16:0 OMPT, it is likely that 16:0 OMPT is also capable of forming chelating micelles in the presence of divalent cations. In 1968 it was reported that EDTA reversed ampicillin resistance in P. aeruginosa; the minimum inhibitory concentrations (MIC) of ampicillin was reduced from 0.5mg/mL to less than 2 microgram/mL in the presence of 3.4 mM EDTA [bib_ref] In vitro reversal of antibiotic resistance by ethylenediamine tetraacetic acid, Weiser [/bib_ref]. The lipopolysaccharide (LPS) layer in Gram negative bacteria such as P. aeruginosa are stabilised by divalent cations (mainly Ca 2+ ). If calcium ions are removed by treatment with a chelator, LPS is liberated, and through this disruption the membrane becomes more permeable to other agents, causing a potentiating action (reviewed in [bib_ref] The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa, Lambert [/bib_ref]. Instead of LPS, Gram positive bacteria have surface sugars, phosphate groups and basic residues organised as teichoic acid. S. aureus teichoic acids are composed of alternating phosphate and ribitol or glycerol groups, and modified with D-alanine and N-acetylglucosamine. Teichoic acids are important for binding of Mg 2+ to the S. aureus cell wall [bib_ref] Teichoic acids and membrane function in bacteria, Heptinstall [/bib_ref] and possibly other divalent cations such as Ca 2+ . The cell surface of S. aureus has a moderately negative net charge at neutral pH, which is probably due to the fact that the teichoic acid contain less positively charged Dalanine residues than negatively charged [bib_ref] Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins,..., Peschel [/bib_ref]. The charge of teichoic acid has been reported to be important for primary adhesion of bacteria to a substrate surface [bib_ref] Key role of teichoic acid net charge in Staphylococcus aureus colonization of..., Gross [/bib_ref] , which is the initial step of biofilm formation. Moreover, the negative charge have been reported to play a role for resistance against glycopeptide antibiotics [bib_ref] The D-alanine residues of Staphylococcus aureus teichoic acids alter the susceptibility to..., Peschel [/bib_ref]. It is thus possible that the chelating ability of 16:0 OMPT and 16:0 MPPA is important for their antibacterial activity through membrane destabilisation. On the other hand, chelators could also cause simple growth inhibition by depriving the bacteria of essential cations. To investigate this we performed flow cytometry analyses [fig_ref] Fig 7: Flow cytometry of MRSA stained with PI [/fig_ref] , which revealed that a high proportion of the cultures treated with 16:0 MPPA and 16:0 OMPT stained positive with PI suggesting that the two compounds directly kill the bacteria, and not just reduce proliferation. Interestingly we found that the while the effect on growth of 16:0 MPPA and EDTA was abolished by addition of 2 mM Ca 2+ , 16:0 OMPT still inhibited growth in the presence of extra calcium [fig_ref] Fig 6: Growth of MRSA in response to lysophosphatidic acids and the influence of... [/fig_ref]. The antibacterial and osteoblast maturation enhancing activity of the LPA (analogue) compounds lead us to investigate bacterial adhesion to 16:0 MPPA and 16:0 OMPT functionalised Ti discs. S. aureus adhesion to the functionalised Ti discs were significantly reduced, however, growth in the media above the functionalised Ti discs were not affected [fig_ref] Fig 8: Lysophosphatidiate-functionalised titanium inhibits MRSA adhesion [/fig_ref]. Moreover, the antiadhesive effect was not reduced in experiments with reuse of functionalised Ti discs. Thus, it seems that only very low or no 16:0 MPPA or 16:0 OMPT were released from the Ti discs into the media. As we found that addition of extra calcium abolished the growth inhibitory effect of 16:0 MPPA, we also investigated the effect of adding additional calcium to the media in which the functionalised Ti discs were placed. However the extra added calcium did not alter bacterial adhesion to neither 16:0 MPPA nor 16:0 OMPT-functionalised surfaces. We also performed SEM on functionalised Ti discs with adherent MRSA bacteria. In accordance with findings obtained using the CFU method, we found that significantly fewer bacteria adhered to the 16:0 OMPT and 16:0 MPPA functionalised Ti compared to the control. The SEM images revealed an interesting and clear difference in morphology between the bacteria adhering to the control, which had a very uneven and coarse surface, and bacteria adhering the 16:0 OMPT and especially the 16:0 MPPA functionalised Ti discs, which appeared smooth and with less extracellular matrix material. This suggests that 16:0 OMPT and 16:0 MPPA affect the teichoic acid layer, destabilising the bacterial outer membrane and thereby affecting both surface attachment and viability. Whilst our findings offer a novel route to the biological modification of Ti we are cognisant that considerably more research will be required to optimise the surface coating. In addition steps to ascertain coating stability and robustness will be priority areas in realising the application of this technology for bone regenerative applications. Specifically an assessment of coating persistence to washing, sterilisation and the physical forces encountered during implantation will need to be part of a wider research plan prior to any in vivo evaluation in the future. # Conclusion To summarise we present evidence for the successful bio-functionalisation of Ti with 16:0 MPPA/OMPT by adopting the facile pre-attachment of an APA via the natural oxide layer of Ti. This surface finish exhibited two noteworthy properties that may help realise the application of selected LPAs in future bone biomaterial design; in the first instance the surfaces enhanced 1,25D-induced hOB maturation. In addition this same surface deterred the attachment of S.aureus and there were noticeable differences in bacterial surface morphology pointing to the possible loss of teichoic acid. Collectively our findings point towards the development of a "dual action" Ti device of which similar technologies have yet to be reported. [fig] Fig 2: Growth and maturation of human (MG63) osteoblasts in response to 16:0 OMPT and 1,25D. A. MG63 osteoblasts were treated with 1,25D (100nM), 16:0 OMPT (micromolarμM) or a combination of these agents and left for three days under conventional cell culturing conditions prior to an assessment of cell growth. As anticipated for the pro-differentiating effects of 1,25D, there were fewer cells (*p<0.01 1,25D versus control) at the end of the culture period. In contrast the application of 16:0 OMPT led to a significant increase in cell numbers (**p<0.01 16:0 OMPT versus control). B. Alkaline phosphatase (ALP) is expressed in greater abundance as osteoblasts progress from an immature to a more differentiated phenotype. Enzyme activity is reliably monitored via the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol (p-NP). The co-stimulation of MG63 cells with 100nM 1,25D and 16:0 OMPT yielded significant increases in total ALP activity by 72 h (*p < 0.001 versus agents in isolation). In each case the data shown are the mean + SD of 4 replicate samples and are a representative from three independent experiments. doi:10.1371/journal.pone.0143509.g002 [/fig] [fig] Fig 3: 16:0 OMPT-functionalised Ti enhances 1,25D-induced MG63 cell maturation. Solid Ti discs (48 in total [/fig] [fig] Fig 4: Lysophosphatidic acid species inhibit the growth of MRSA in Luria broth. The effect of different concentrations of 16:0 MPPA, 16:0 OMPT and EDTA on growth of MRSA 43484 in Luria broth. Legend: 16:0 MPPA; black (control), red (1,56 microgram/mL), green (6,3 microgram/mL), blue (25 microgram/mL). 16:0 OMPT; black (control), red (1,56 microgram/mL), green (6,3 microgram/mL), blue (25 microgram/mL). EDTA; black (control), green (6,3 microgram/mL), blue (25 microgram/mL), grey (0.1mg/mL). doi:10.1371/journal.pone.0143509.g004 [/fig] [fig] Fig 5: Lysophosphatidic acid species inhibit the growth of MRSA in minimal media. The effect of different concentrations of 16:0 MPPA, 16:0 OMPT and EDTA on growth of MRSA 43484 in minimal media. Legend: 16:0 MPPA; black (control), red (1,56 microgram/mL), green (6,3 microgram/mL), blue (25 microgram/mL). 16:0 OMPT; black (control), red (1,56 microgram/mL), green (6,3 microgram/mL), blue (25 microgram/mL). Notice that the symbols of the control almost hides the symbols of 6,3 microgram/mL EDTA. doi:10.1371/journal.pone.0143509.g005 [/fig] [fig] Fig 6: Growth of MRSA in response to lysophosphatidic acids and the influence of calcium. Growth curves of MRSA 43484 in minimal media showing the effect of Ca 2+ . Black (control), red (control + 2 mM Ca 2+ ), green (100 μg/mL 16:0 MPPA/16:0 OMPT/EDTA), Blue 0.1mg/mL 16:0 MPPA/16:0 OMPT/ EDTA + 2 mM Ca 2+ ). doi:10.1371/journal.pone.0143509.g006 [/fig] [fig] Fig 7: Flow cytometry of MRSA stained with PI: effect of lysophosphatidic acid species and EDTA on viability. Histograms of MRSA 43484 stained with propidium iodide (PI), gated on forward and side scatter. Bacteria were grown in the presence of 0.1mg/mL 16:0 MPPA, 50 microgram/mL 16:0 OMPT, 0.1mg/mL EDTA or in the absence of compounds (control). Markers show the percentage of PI stained, and hence nonviable, population. doi:10.1371/journal.pone.0143509.g007 MPPA and/or 16:0 OMPT. Fig 8C shows that adding 2 mM Ca 2+ did not affect the anti-adhesive properties of 16:0 MPPA or 16:0 OMPT functionalisation. [/fig] [fig] Fig 8: Lysophosphatidiate-functionalised titanium inhibits MRSA adhesion. Effect of functionalised Ti on bacterial adhesion. A-C) CFU of adherent MRSA pr. Ti disc after detachment. A) CFU pr. Ti disc; control or functionalised with either 16:0 OMPT or 16:0 MPPA. B) *Reuse of the same discs as in A) after washing and autoclaving. C) Effect of addition of 2 mM Ca 2+ on MRSA adhesion to Ti discs. In each instance four discs were used for each of the different Ti surface treatments. D-F) End point OD 450 of planktonic bacteria in the wells containing the Ti discs in A-C. (*p<0.05, **p<0.005 and ***p<0.0005 compared to control). Each of the figures is a representative of four independent experiments. doi:10.1371/journal.pone.0143509.g008 [/fig] [fig] Fig 9: Lysophosphatidate-functionalisation of titanium deters bacterial attachment and alters bacterial surface morphology. A. Scanning Electron Microscopy images of MRSA 43484 on control and functionalised Ti surfaces. The left column shows unmodified Ti, in the centre, Ti functionalised with 16:0 OMPT, and in the right column images of bacteria upon 16:0 MPPA functionalised Ti. B. Graph depicting the data obtained for the number of bacteria associated for each of the different Ti surfaces. doi:10.1371/journal.pone.0143509.g009 [/fig]

The spike glycoprotein of SARS-CoV-2: A review of how mutations of spike glycoproteins have driven the emergence of variants with high transmissibility and immune escape [bib_ref] Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus, Sevajol [/bib_ref] [bib_ref] Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading..., Smith [/bib_ref] [bib_ref] Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Wu [/bib_ref] [bib_ref] Epidemic dynamics and antigenic evolution in a single season of influenza A, Boni [/bib_ref] [bib_ref] Maximum Daily Temperature, Precipitation, Ultraviolet Light, and Rates of Transmission of Severe..., Sehra [/bib_ref] ## Could vocs overshadow the breakthrough of vaccines? To have an overview of the impact of mutations that generate VoCs on vaccine efficacy as revealed by experiments with SARS-CoV-2 itself or pseudoviruses expressing combined or individual mutations on S protein that led to VoCs, sera of 20 post-vaccinated volunteers immunized with BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna), both mRNA vaccines, presented levels of anti-spike IgM and IgG and RBD-specific antibodies similar to people that were naturally infected by SARS-CoV-2. The results revealed a reduction in the neutralization of SARS-CoV-2 or pseudoviruses carrying mutations compared to the Wuhan isolate. In another experiment, the effect was evaluated of a single N501Y mutation on neutralization. Results showed that sera from 20 participants in a trial of BNT162b2 presented a modest reduction in the neutralization of virus, with N501Y mutation [bib_ref] Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera, Xie [/bib_ref] [bib_ref] Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2..., Xie [/bib_ref]. COVID-19 patients, modulates glycosylation at the nearby N616 site, but has higher infectivity and transmissibility of SARS-CoV-2 [bib_ref] Will SARS-CoV-2 variants of concern affect the promise of vaccines?, Gupta [/bib_ref] [bib_ref] Functional importance of the D614G mutation in the SARS-CoV-2 spike protein, Jackson [/bib_ref]. At the molecular level, the D614G mutation increases the stability and density of the S protein. It decreases the S1 subunit shedding, keeping both S1 and S2 subunits more closely supported by stronger intermolecular association, leading to a highly stabilized S1-S2 interface in SD614G. It was known that S1 shedding limits the infectivity of the original SARS-CoV-2 (without SD614G) by releasing S1 from S2 during viral fusion. The D614G mutation fixed this problem by strengthening the intramolecular interaction between S1 and S2, blocking S1 dissociation and thus virus infectivity and transmission [bib_ref] Will SARS-CoV-2 variants of concern affect the promise of vaccines?, Gupta [/bib_ref] [bib_ref] Functional importance of the D614G mutation in the SARS-CoV-2 spike protein, Jackson [/bib_ref]. ## Alpha variant of sars-cov-2 In December 2020, a variant that spread quickly and increased the risk of death was detected in the United Kingdom (UK). The variant derived from the SARS-CoV-2 20B clade was confirmed as B.1.1.7, now known as the Alpha variant. In the early investigation stage, increased transmissibility of up to 71% over the previously circulating strains of SARS-CoV-2 [bib_ref] New variant of SARS-CoV-2 in UK causes surge of COVID-19, Kirby [/bib_ref] [bib_ref] Emergence of a new SARS-CoV-2 variant in the UK, Tang [/bib_ref] was estimated. The high transmissibility of the Alpha variant has been described in several studies in different countries, with a transmissibility range between 50-80% [bib_ref] Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the..., Leung [/bib_ref]. In vitro and in vivo experiments supported the observed behavior of the Alpha variant. It was demonstrated that the Alpha variant can multiply and shed more effectively in the nasal cavity of hamsters than other variants, even at low viral loads and short time of exposure [bib_ref] Low dose inocula of SARS-CoV-2 Alpha variant transmits more efficiently than earlier..., Mok [/bib_ref]. Based on that, the Alpha variant was defined as VoC in December 2020. It was later defined as a variant, being monitored (VBM) because it had no longer been detected or was circulating at very low levels. ## J o u r n a l p r e -p r o o f In January 2021, 45 countries reported the Alpha variant's presence. A group of British scientists proposed that the Alpha variant is responsible for the third wave of the pandemic in Scotland, associated with severe cases of COVID-19. The study results showed that the Alpha variant of SARS-CoV-2 has 5 times higher permeability than other co-circulating variants, hence its rapid spread in Scotland. In a UK case-control study of 54,906 participants who tested positive for SARS-CoV-2, in comparison with the Alpha variant and previously circulating variants reported a mortality hazard ratio associated with infection with VOC-202012/1 compared with infection with previously circulating variants (hazard ratio of 1.64 [95% CI 1.32 -2.04]) [bib_ref] Risk of mortality in patients infected with SARS-CoV-2 variant of concern 202012/1:..., Challen [/bib_ref]. The Biwako Ohashi Hospital in Japan described a specific sign called red face after Alpha variant infection, which may be predictive of a sudden deterioration of patients [bib_ref] Red face may be a specific sign of SARS-CoV-2 Alpha variantAlpha v\, Nakaya [/bib_ref]. Interestingly, in September 2021, a French group had already identified the SARS-CoV-2 Alpha variant in cats and dogs in France [bib_ref] Report of One-Year Prospective Surveillance of SARS-CoV-2 in Dogs and Cats in..., Krafft [/bib_ref]. Recently, scientists in the UK identified cases of severe myocarditis in cats and dogs associated with the Alpha variant that crossed the interspecies line in the that country, raising questions regarding its potential pathogenicity in these animals [bib_ref] Infection with SARS-CoV-2 variant B.1.1.7 detected in a group of dogs and..., Ferasin [/bib_ref]. The Alpha variant results from the accumulation of 17 mutations compared to the original SARS-CoV-2 virus discovered in Wuhan, China. Here, as reported before, we focus on the mutations accumulated in the S protein [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. These mutations are expected to lead to expanded spread and increased severity of the associated disease, requiring more effective vaccines, therapeutic drugs, diagnostic tools, and other public health measures. The Alpha variant has eight spike mutations, including two deletions in the S protein [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. These mutations led to two deletions, both in the NDT region: 1) 69-70del (i.e., a deletion of 6 bases coding for histidine and valine at positions 69 and 70, respectively, in the E484K, N501Y, and A701V). Three further spike mutations emerged by the end of November (L18F, R246I, and K417N). Additionally, the Beta variant has two deletions in the NDT region [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. The substitutions are: L18F, involving the exchange of a leucine (apolar amino acid) by a phenylalanine (a hydrophobic amino acid); D80A, the replacement of aspartic acid (negative) with alanine (apolar); D215G, replacement of an aspartic acid (negative) with a glycine (apolar); and R246I, involving replacement of an arginine (positive) with an isoleucine (apolar). Also, deletions in the NDT region [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref] are involved in the escape from neutralization by antibodies such as NTD-binding mAbs30 and 4A8. Three of the spike mutations are in critical residues in the receptor-binding domain (RBD) (K417K, E484K, and N501Y) [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. The K417 position of the S protein has been designated an epitope of RBD of class 1 and 2 antibodies. Moreover, very likely the alteration of this spot affects the ability of class 1 and 2 antibodies to bind and neutralize the RBD, preventing the interaction with the ACE2 receptor. In the Beta variant, a substitution of lysine (positive) by asparagine (polar uncharged) is involved in the ability of the S protein from the Beta variant to escape from immune antibodies. Besides that, this substitution has a negative effect. It was noticed that K417N slightly reduces the affinity of RBD for ACE2, but it seems this collateral effect did not change the Beta variant's fitness. The substations E484K and on RBD possibly enhanced the binding affinity of the Beta variant for the ACE2 receptor. The E484K mutation is uncommon and interacts with the K31 hotspot residue of hACE2 [bib_ref] Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants, Weisblum [/bib_ref]. The position E484 of the S protein indicates it is an immunodominant residue, which means the immune response is focused against it. So, mutation J o u r n a l P r e -p r o o f in this position could completely disarm the immune response toward a variant holding it. The Beta variant was the first VoC to show substitution at position 484, in which a proline (apolar) was replaced by lysine (positive). The E484K is involved in escape from antibodies in convalescent plasma (C121 and C144 monoclonal antibodies) [bib_ref] Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants, Weisblum [/bib_ref] [bib_ref] Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization, Liu [/bib_ref] [bib_ref] SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma, Andreano [/bib_ref] [bib_ref] Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with..., Baum [/bib_ref]. Baum et al. [bib_ref] Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with..., Baum [/bib_ref] showed that a pseudovirus with the mutation E484K could escape neutralization by combining two monoclonal antibodies, REGN10989 and REGN10934, used to treat severe cases of COVID-19. The mutation N501Y (see the Alpha variant section) is present in Alpha and Beta. It is involved in the binding phase of the S protein with ACE2, allowing an increase in affinity for ACE2 and modifying the structural mechanics of the RBD-ACE2 complex. The N501Y enables escape from the immune system but reduces the affinity of the S protein for the ACE2 receptor (see the Alpha variant section). However, in the Beta variant, the presence of K417N and E484K mutations slightly reduce the affinity for RBD, as they abolish the interfacial salt bridges that help bind RBD-ACE2 and stabilize the complex. Therefore, these two additional mutations in the Beta variant could overshadow in part the increase in the RBD-ACE2 caused by N501Y. However, those mutations facilitate the escape of the Beta variant from the neutralizing effect of antibodies. Based on that, it is feasible to suggest that the positive effect of K417N and E484K mutations is greater than the negative effect, enhancing the Beta variant's fitness and thus being kept by the virus and even developed by other variants [bib_ref] Timing the SARS-CoV-2 index case in Hubei province, Pekar [/bib_ref] [bib_ref] Temporal signal and the phylodynamic threshold of SARS-CoV-2, Duchene [/bib_ref]. As discussed above, the Beta variant presents many non-synonymous amino acid substitutions. These amino acid changes alter the three-dimensional structure of the S protein. For example, N501Y replaces an asparagine with a tyrosine. The side-chain volume of asparagine is 114.1 Å 3 , compared to the side-chain volume of 193.6 Å 3 of tyrosine. This J o u r n a l P r e -p r o o f difference in the size of lateral chains affects the S protein's structure. To verify that, a root mean square deviation (RMSD) calculation of the atomic position on the S protein from the variants was performed in comparison with the wild-type S protein from the Wuhan isolate [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. To perform the structural alignments, the 3D structures of S proteins of the Wuhan isolate (PDB ID: 6Z97) were deposited in the Protein Data Bank (PDB, https://www.rcsb.org/) and the database of the Beta variant (modeled in this study). The RMSD analysis revealed significant structural changes between Wuhan S protein and Beta variant S protein [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. It is possible to analyze in many positions the differences of the two proteins. The RMSD value for this alignment was 1.604 A, indicating that both atomic positions are quite different. The greatest alteration was in regions such as NDT, the arm of S1, and RBD [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. These alterations are responsible for the differential interaction of the S protein with the ACE2 receptor, and thus the spread, infectivity, and severity of the disease. Given the ability to escape from the immune system, the Beta variant overtook the Alpha [bib_ref] Emergence and spread of a SARS-CoV-2 lineage A variant (A.23.1) with altered..., Bugembe [/bib_ref] [bib_ref] Temporal signal and the phylodynamic threshold of SARS-CoV-2, Duchene [/bib_ref]. Experiments using viruses expressing RBD with K417N + E484K + N501Y revealed a reduction of 2 to 3-fold in neutralization by antibodies in plasma from vaccinated individuals. J o u r n a l P r e -p r o o f ## Gama variant of sars-cov-2 Since SARS-CoV-2 became pandemic, it has evolved genetically, leading to different variants [fig_ref] Figure 1: Scheme model showing all the mutation on Spike protein from variants [/fig_ref] , with different genetic profiles, and thus different degrees of severity. The genomic sequencing of viral samples has been fundamental to detect the new variants of SARS-CoV-2 [bib_ref] Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the..., Korber [/bib_ref]. The new VoC, the P.1 lineage, known today as the Gama variant, was detected in about 42% of the analyzed genomic sequences in samples from Manaus in December 2020 and presented important mutations with biological significance. The Gamma variant was also identified in January 2021 in Brazilian travelers at Japanese airports [bib_ref] Discovery of a SARS-CoV-2 variant from the P. 1 lineage harboring K417T/E484K/N501Y..., Hirotsu [/bib_ref]. After that, this variant was identified in different Brazilian regions [bib_ref] The emergence of novel SARS-CoV-2 variant P. 1 in Amazonas (Brazil) was..., Freitas [/bib_ref] [bib_ref] New Brazilian variant of the SARS-CoV-2 (P1) of COVID-19 in Alagoas state, Silva [/bib_ref]. Manaus, the capital of Amazonas State, was the most affected and became an epicenter of the outbreak of the Gamma variant, which quickly spread through the Solimões River course, causing the collapse of public and private health systems [bib_ref] COVID-19 in Amazonas, Brazil, was driven by the persistence of endemic lineages..., Naveca [/bib_ref]. Notably, the cases of Gamma variant in January represented 85.4% (41/48) of COVID-19 cases in Manaus. In February 2021, it had spread in South America and was responsible for nearly 40% of COVID-19 cases [bib_ref] Novel SARS-CoV-2 variants: the pandemics within the pandemic, Boehm [/bib_ref] [bib_ref] Rapid Emergence and Spread of Severe Acute Respiratory Syndrome Coronavirus 2 Gamma..., Tagliamonte [/bib_ref]. These results revealed the high infectivity rate of this variant, which is estimated to be 2.6 times more infectious. In February 2021, two cases of a new SARS-CoV-2 variant were described in Alagoas State. One person came from Amazonas, and the other was characterized as community transmission in Alagoas, confirming the presence of the Gamma variant [bib_ref] New Brazilian variant of the SARS-CoV-2 (P1) of COVID-19 in Alagoas state, Silva [/bib_ref]. According to data gathered by the FIOCRUZ-COVID-19 Genomics Surveillance Network of the Brazilian Ministry of Health, the Gamma variant was mainly responsible for Covid-19 cases in Brazil from February to July 2021. For comparison, in December 2020, 26% of deposited genomes were the Gamma variant (P.1); six months later, in June, 64.6% were the Gamma variant, and in August, 99% of the sequenced samples were the Gamma variant [bib_ref] A vigilância genômica do SARS-CoV-2 no Brasil na resposta à pandemia da..., Sallas [/bib_ref]. After the first detection on 23 December 2020 in Manaus, the Gamma variant was found in 71 countries. It was detected in South America, Central America, North America, Europe, Angola, Turkey, Japan, the Philippines and Australia. The Gamma variant was not detected following a logical geographical sequence concerning the first case, since it was detected in Uruguay, Canada, India and Australia in the same period. Brazil and the United States had the greatest number of Gamma infections globally [bib_ref] Immunity to SARS-CoV-2 variants of concern, Altmann [/bib_ref]. The Gamma variant was classified as VoC based on its greater transmissibility, pathogenicity, and immune escape. The Gamma variant carries multiple mutations in the NDT and RBD domains [bib_ref] Novel SARS-CoV-2 variant in travelers from Brazil to Japan, Fujino [/bib_ref] [bib_ref] Genomic evidence of SARS-CoV-2 reinfection involving E484K spike mutation, Brazil, Emerg, Nonaka [/bib_ref]. The analysis of sequences revealed that the SARS-CoV-2 Gamma variant has 37 mutations, including 22 missense, 10 synonymous, three intergenic, one frameshift, and one inframeshift mutation. In the spike protein, 12 missense mutations (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F) were observed [bib_ref] Genomic evidence of SARS-CoV-2 reinfection involving E484K spike mutation, Brazil, Emerg, Nonaka [/bib_ref] [bib_ref] First Identified Cases of SARS-CoV-2 Variant P. 1 in the United States-Minnesota, Firestone [/bib_ref]. Three mutations (K417T, E484K, and N501Y) were also identified in the RBD region of the spike protein [bib_ref] Discovery of a SARS-CoV-2 variant from the P. 1 lineage harboring K417T/E484K/N501Y..., Hirotsu [/bib_ref] [bib_ref] Novel SARS-CoV-2 variant in travelers from Brazil to Japan, Fujino [/bib_ref]. (replacing a threonine with an asparagine) and P26S (replacing a proline with a serine) are located in the NDT supersite region. The NDT supersite is highly targeted by antibodies [bib_ref] N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2, Mccallum [/bib_ref]. Studies by McCallum et al. [bib_ref] N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2, Mccallum [/bib_ref] and Suryadevara et al. [bib_ref] Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the..., Suryadevara [/bib_ref] revealed that antibodies with higher neutralizing power preferably attack the NDT supersite region. Thus, a mutation in this region helps the Gamma variant to escape from the immune system. Additionally, the mutation T20N provides an N site for glycosylation, which could create a shield, protecting the region from antibody recognition. Although D138Y, R190S, H655Y, and T1027I are non-synonymous mutations, it seems that their mutations have not affected or conferred any additional fitness to the S protein, or at best, their effects are still unclear. What is known is that those mutations are lineage-defining. RMSD analysis comparing the S protein from the Gamma variant with the S protein from the Wuhan isolate produced a score of 1.060 Å, indicating the differences between S proteins [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. Compared to other variants, the S protein from the Gamma variant is the one closest to the S protein from the Wuhan isolate. The most exciting alterations in the 3D structure are in the NDT region [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref] , and the mutation T20N, which potentially increases the glycosylation, covering the entire region, preventing the antibodies' attachment. The RMSD analysis suggests that the S protein from the Gamma variant has adapted its structure mainly to J o u r n a l P r e -p r o o f escape from the immune system. The Gamma variant is responsible for the first case of reinfection in Manaus, Brazil. This observation indicates the Gamma variant has excellent ability to mitigate or escape from the immune system, which might be related to a higher mortality rate of the Gamma variant, estimated to be 1.7 to 2.4-fold higher than the rate of the Wuhan isolate. The Gamma variant has a high attack rate even in people that have completed the vaccination regimen. By July 2021, the Gamma variant was predominant in French Guiana, threatening the hospital capacity by causing the third wave [bib_ref] Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other..., Letko [/bib_ref]. The reduced vaccine efficiency towards the Gamma variant was a surprise, because in vitro trials showed strong neutralization of antibodies in people vaccinated with the BNT162b2 vaccine [bib_ref] Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine, Polack [/bib_ref] [bib_ref] Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera, Xie [/bib_ref] [bib_ref] Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2..., Xie [/bib_ref] [bib_ref] Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity, Garcia-Beltran [/bib_ref]. The unexpectedly high attack rate of the Gamma variant could result from problems with the vaccine storage, but this is just speculation. A Gamma outbreak among persons fully vaccinated with the BNT162b2 vaccine shows the ability of the Gamma variant to escape from the immune system. This uncommon infection of the Gamma variant in vaccinated people demands surveillance and studies of BNT162b2's effectiveness in regard to the Gamma variant. ## Delta variant First reported in India, in late December 2020, the double-mutated Delta VoC, a coronavirus variant formerly known as Indian VoC or B.1.617.2, is 40% more transmissible than many countries in Europe have faced increased new infections and deaths by the Delta variant, which rapidly became the dominant variant worldwide [bib_ref] Delta to dominate world, Vaughan [/bib_ref]. The advantage of this Delta variant is the combination of high transmissibility and immune system evasion, besides being less sensitive to neutralizing antibodies. Mutations of the S protein may have enhanced its ability to bind to the ACE2 receptor [bib_ref] SARS-CoV-2 Delta VOC in Scotland: demographics, risk of hospital admission, and vaccine..., Sheikh [/bib_ref]. As discussed above, the S protein must be cut twice by host proteins to enter human cells. However, the Delta variant has earlier processing of the S protein, and newly formed viral particles emerge rapidly from infected cells, infecting other cells more efficiently [bib_ref] The mutation that helps Delta spread like wildfire, Callaway [/bib_ref]. The Delta variant has accumulated multiple mutations in the S1 subunit, specifically in the NDT and RBD regions. Mutations in NDT are related to escape from the immune system, and mutation in RBD seems to improve its stability and ability to bind to ACE2 and evade the immune system [bib_ref] Emerging SARS-CoV-2 variants of concern and potential intervention approaches, Khateeb [/bib_ref]. However, despite this knowledge, many unknowns surround the Delta variant. For instance, it is not yet understood for sure how Delta's mutations lead to a supercharged variant [bib_ref] Emerging SARS-CoV-2 variants of concern and potential intervention approaches, Khateeb [/bib_ref]. The mutations T19R, V70F*, T95I, G142D, delE156-, delF157-, R158G, A222V* and W258L* are all located in the NDT [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref] region, covering the NDT supersite, which is the target of anti-NTD neutralizing antibodies [bib_ref] Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity, Garcia-Beltran [/bib_ref] [bib_ref] N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2, Mccallum [/bib_ref]. The NDT region has a stabilizing effect on the S protein [bib_ref] A neutralizing human antibody binds to the N-terminal domain of the Spike..., Chi [/bib_ref] because it is targeted by potent neutralizing antibodies [bib_ref] N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2, Mccallum [/bib_ref] [bib_ref] Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the..., Suryadevara [/bib_ref] [bib_ref] A neutralizing human antibody binds to the N-terminal domain of the Spike..., Chi [/bib_ref] [bib_ref] Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single..., Cerutti [/bib_ref] [bib_ref] Potential covalent drugs targeting the main protease of the SARS-CoV-2 coronavirus, Liu [/bib_ref]. The NDT supersite is divided into five loops, named N1 to N5, covering different residue gaps in N1 (residues 14-26), N2 (residues 67-79), N3 (residues 141-156), N4 (residues 177-186), and N5 (residues 246-260) [bib_ref] A neutralizing human antibody binds to the N-terminal domain of the Spike..., Chi [/bib_ref] [bib_ref] Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single..., Cerutti [/bib_ref] [bib_ref] An NTD supersite of attack, Lok [/bib_ref]. The Delta variant has accumulated many non-synonymous mutations in the NDT supersite, changing its structure to reduce the neutralizing effect of anti-NDT antibodies. A meticulous analysis of the Delta variant's mutations in the NDT region shows that T19R is in the N1 region, V70F is in the N2 region, G142D and delE156-are in N3, and A222V and W258L are in the N5 region. The Delta variant showed three mutations in the RBD region (K417N, L452R, T478K). One of them, K417N, shared with the Beta and Gamma variants, has already been discussed. Here we focus on the two exclusive regions of the Delta variants L452R and T478K [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. It was shown that the Alpha, Beta, and Gamma variants share the mutation N501Y, which is involved in escaping from the immune system. However, this mutation slightly reduces the affinity of RBD for the ACE receptor. It seems that the Delta variant has accumulated mutations to overcome that obstacle. The Delta variant lacks the mutation N501Y. In contrast, it has the mutations L452R and T478K, which are involved in the escape from anti-RBD antibodies and do not reduce the affinity of RBD for ACE2 [bib_ref] On the origin and continuing evolution of SARS-CoV-2, Tang [/bib_ref] [bib_ref] Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies, Li [/bib_ref] [bib_ref] N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2, Mccallum [/bib_ref] [bib_ref] A neutralizing human antibody binds to the N-terminal domain of the Spike..., Chi [/bib_ref] [bib_ref] Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single..., Cerutti [/bib_ref] [bib_ref] A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2..., Yuan [/bib_ref] [bib_ref] Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor..., Lu [/bib_ref]. The mutation L452R (changing a leucine for an arginine) is closely related to the escape of monoclonal antibodies. For example, the monoclonal antibody Bamlanivimab lost its J o u r n a l P r e -p r o o f antiviral effect against the Delta variant, contrasting with its continuing activity against other variants. The unique change of T478K in the RBD region of the Delta S protein was found precisely in the epitope region recognized by monoclonal antibodies known as Class 1. The mutation P681R, adjacent to the furin cleavage site (685), seems to be one of the probably is the essential advantage gained by Delta [bib_ref] The mutation that helps Delta spread like wildfire, Callaway [/bib_ref]. Liu and researchers reported that the S protein is cut more efficiently in the Delta variant than others [bib_ref] Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant, Liu [/bib_ref]. The P681R at a furin cleavage site separates the S1 and S2 subunits [bib_ref] The mutation that helps Delta spread like wildfire, Callaway [/bib_ref]. The authors stated that it seems the viral particles of the Delta variant are produced with the primed S protein and then infect cells more efficiently. Virologists at the University of Tokyo showed that the P681R mutation speeds up the infection of uninfected cells through plasma membrane fusion, three times more than the other variants. ## J o u r n a l p r e -p r o o f journal pre-proof The D950N [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref] region is located at the interface of the S protein trimer. It is suggested that this mutation regulates S protein dynamics between monomeric and trimeric phases [bib_ref] The variant gambit: COVID-19's next move, Plante [/bib_ref]. The RMSD analysis [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref] revealed that the S protein from Delta is the most different from the original S protein from the Wuhan isolate [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. The S protein from the Delta variant presented the highest value of RMSD, 1.716 [fig_ref] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. The most altered region is the NDT region due to accumulating a higher number of mutations. The Delta variant is more aggressive [bib_ref] Outbreak of Coronavirus (SARS-CoV-2) Delta variant (B.1.617.2) and Delta Plus (AY. 1)..., Malabadi [/bib_ref]. Delta-infected people present a viral load 1,260 times higher than people infected with the original coronavirus strain [bib_ref] How the Delta variant achieves its ultrafast spread, Reardon [/bib_ref]. Li et al.reported that high viral loads in people infected with the Delta variant are reached in up to four days after contact with an infected person. In contrast, in people infected with the original SARS-CoV-2, this only happened six days after contact. This result indicates a higher replication rate of the Delta variant, which has a perfect combination to spread. The high viral loads plus short incubation time make sense to explain the greater transmissibility of the Delta variant compared to the original SARS-CoV-2 or even the other variants. The Delta variant's ability to strongly bind to the receptors of lung cells and escape from immune response might overcome immunization gained from vaccination [bib_ref] Outbreak of Coronavirus (SARS-CoV-2) Delta variant (B.1.617.2) and Delta Plus (AY. 1)..., Malabadi [/bib_ref]. Recent data indicate that fully vaccinated people can still be vectors to spread the Delta variant more efficiently than other variants [bib_ref] How do vaccinated people spread Delta? What the science says, Subbaraman [/bib_ref]. The fully vaccinated people are protected against severe COVID-19 and death by Delta variants. However, those people still present a high level of transmission. This fact is intriguing to scientists worldwide, and the recommendation is for vaccinated people to continue taking precautions such as social distancing and wearing face masks. ## J o u r n a l p r e -p r o o f ## Omicron variant On 24 November 2021, a new variant was identified in South Africa, variant B.1.1.529, later designated as Omicron by the WHO. Two days later, Omicron was classified as a VoC, a classification that occurred faster than the other variants. The Omicron variant is highly mutated, with more than 50 mutations (30 mutations in the S protein alone). There have been more than 200 cases detected in 23 countries on all 6 continents. Most surprisingly, cases were detected in Scotland, where 6 cases of these variants were identified. None of them were related to travelers from South Africa, which may indicate community transmission of the variant [bib_ref] Heavily mutated coronavirus variant puts scientists on alert, Callaway [/bib_ref]. Initial results show that this variant has many mutations in relation to the Alpha and Delta variants, linked to the sudden increase in transmission, infectivity, and escape from the immune system. Initial modeling studies using artificial intelligence showed that Omicron´s protein S could escape from T lymphocyte cells, but more in vitro studies need to be conducted to confirm this finding [bib_ref] Heavily mutated coronavirus variant puts scientists on alert, Callaway [/bib_ref]. Like three patients in Brazil, some people infected by Omicron were fully vaccinated, indicating that Omicron can defeat the immunity caused by vaccines. Preliminary studies have shown that the S protein of this new variant has an insertion at position 214 of the NTD region. This insertion was likely acquired from a host infected by SARS-CoV-2. Therefore, sequencing in the search for new variants is of paramount importance for understanding how mutations affect the behavior of protein S in human infection and the discovery of new animal hosts. That is why it is essential to continue monitoring people infected with SARS-CoV-2. ## J o u r n a l p r e -p r o o f journal pre-proof Of the 30 mutations of the S protein of the Omicron variant, some are shared with previous variants, and others are unique. Among the mutations, Omicron has one insertion of three amino acids in the NDT region together with three small deletions compared to wild-type SARS-CoV-2 from Wuhan. Insertions and deletions in the S protein from Omicron lead to a loss of three amino acid residues in the Omicron S protein compared to the wild-type version of the virus. Indeed, mutations lead to a different S protein in the Omicron variant compared to S protein from Wuhan SARS-CoV-2 [fig_ref] Figure 5: Structural alignment among Spike protein from Wildtype and variants of SARS-CoV-2 [/fig_ref]. The Delta variants have the most different S protein among the variants compared to Wuhan SARS-CoV-2 [fig_ref] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants... [/fig_ref]. However, RMSD analysis of the S protein from Omicron revealed a new winner. The S protein from the Omicron variant had an RMSD score of 2.156 [fig_ref] Figure 5: Structural alignment among Spike protein from Wildtype and variants of SARS-CoV-2 [/fig_ref] , compared to the S protein structure of Wuhan isolate [fig_ref] Figure 5: Structural alignment among Spike protein from Wildtype and variants of SARS-CoV-2 [/fig_ref] , with score of 1.716 [fig_ref] Figure 5: Structural alignment among Spike protein from Wildtype and variants of SARS-CoV-2 [/fig_ref]. This analysis showed many alterations in atoms' positions, leading to a structural difference in S protein spatial arrangement. The most different parts of S protein from the Omicron variant have been found in the NDT region, which has suffered from many mutations, including insertions and deletions. First of all, the D614G substitution in all variants is also conserved in Omicron S protein. Omicron shares mutations that were previously exclusive to Alpha, such as del69-70 and P681H. [fig_ref] Table 1 Table 2: Data comparison of COVID-19 with other viral diseases Variants of SARS-CoV-2 J... [/fig_ref] , N969K, and L981F [bib_ref] Heavily mutated coronavirus variant puts scientists on alert, Callaway [/bib_ref]. These mutations are found in the NDT region, in the RBD, around the furin cleavage region, in the fusion peptide and HR1 regions. The shared mutation between Omicron and other variants has already been discussed throughout this review. When important, it is possible to further develop some discussion. However, the focus here is on the signature mutations presented by Omicron. An interesting point regarding Omicron is the substitution of E484A rather than the E484K present in the Beta and Gamma variants (Figs. 3A and 4A). As discussed above, the E484K is related to escape from neutralizing antibodies. In the Omicron variant, the E484A is more involved in escaping from 2B04 and 1B07monoclonal antibodies [bib_ref] Heavily mutated coronavirus variant puts scientists on alert, Callaway [/bib_ref]. As happens in other variants, the mutation in the NTD region was mostly involved in two points: stabilizing the S1 to allow strong interaction between RBD and ACE2, and escape from neutralizing antibodies. With the deletion shared with the Alpha variant, Omicron also has another del211, a substitution of L212I, and an ins214EPE of three amino acids in the NDT region. As discussed above, NDT is a supersite of recognition by antibodies. By changing this region, Omicron can be more efficient in escaping from the immune system [bib_ref] Recoding: translational bifurcations in gene expression, Baranov [/bib_ref] [bib_ref] Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape, Mccarthy [/bib_ref]. These alterations could be involved in the ability of Omicron to infect fully vaccinated people. This is not the first substitution where N440K is present. One SARS-CoV-2 sample isolated from a re-infected patient presented this mutation [bib_ref] Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants, Weisblum [/bib_ref] and is related to escape from human monoclonal antibodies. This mutation is predicted to result from selective pressure imposed by antibodies [bib_ref] Symptomatic reinfection of SARS-CoV-2 with spike protein variant N440K associated with immune..., Rani [/bib_ref]. Other mutations in RBD such as G446S, S477N, T478K (also in J o u r n a l P r e -p r o o f the Delta variant), E484A, and Q498R, are all involved in the escape of RBD from anti-RBD neutralizing antibodies [bib_ref] Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant, Liu [/bib_ref] [bib_ref] The spike protein of SARS-CoV-2 variant A.30 is heavily mutated and evades..., Arora [/bib_ref]. Q493 is an important site for interaction between RBD and ACE2. However, mutations at this site are generally either Q493R/K, and the Omicron variant has the Q4 substitution It seems that this mutation is tolerated and does not affect the interaction between ACE2 and RBD [bib_ref] Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555..., Starr [/bib_ref]. Indeed, this mutation is involved in the escape from LY-CoV555, LY-CoV016, and REGN10989/10934 monoclonal antibodies [bib_ref] Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with..., Baum [/bib_ref] [bib_ref] Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555..., Starr [/bib_ref]. S477N, N501Y, P681H, and N679K are very likely to support the Omicron variant's higher transmissibility. Before Delta, Alpha was the most transmissible variant in the world. This was possible because compared to wild-type SARS-CoV-2, the Alpha variant has a histidine rather than a proline at position 681 in the polybasic cleavage site in the spike protein at the S1/S2 junction (residues 681-685) to the furin site of cleavage. The furin cleavage requires a basic environment to occur, so the replacement of proline (apolar) by histidine (positively charged) in the Alpha variant improved the furin cleavage, producing more activity of S proteins able to infect the cell. Additionally, the Alpha variant loses glycosylation at this position by removing the proline residue. This is important because carbohydrates hinder the cleavage site, inhibiting access by the protease. Elimination of proline removed a glycosylation site found in the ancestral Wuhan strain. In the case of Delta, there has been improvement. The Delta variant has substitution at position 681, but the Delta variant has an arginine replacing the proline residue instead of lysine. By being the essential amino acid residue, the Delta variant improved the cleavage of S protein, enhancing its cell recognition and infection, leading to higher transmissibility than that presented by the Alpha variant. The Omicron preserves the same substitution presented by the Alpha J o u r n a l P r e -p r o o f variant, P681H. The replacement of proline by an arginine instead of histidine is a significant evolutionary advantage. In contrast to histidine, which is only positive at mildly basic pH, arginine is positive under all cell conditions. This is thought to be responsible for the higher transmissibility of the Delta variant [bib_ref] Furin cleavage of the SARS-CoV-2 spike is modulated by Oglycosylation, Zhang [/bib_ref]. At first glance it was reasonable to assume that Omicron could be less transmissible than the Delta variant. However, Omicron also showed a new mutation near the cleavage site, N679K. This substitution also added a positively charged lysine residue, making the neighborhood satisfactory for furin cleavage. So, these two mutations, P681H and N679K seem to be associated with mutations that provide a more efficient S1-S2 cleavage, leading to a more infective S protein. Although this makes total sense, it needs more investigation for confirmation. However, the rapid replacement of the Delta variant by Omicron in South Africa reinforces the hypothesis of high transmissibility rates of the Omicron variant. Another explanation for higher transmissibility of Omicron is that these mutations also promote escape from the immune system. There is no doubt about the higher transmissibility rates of Omicron. However, it needs to be quantified. The reproductive number (R 0 ) is a measure of transmissibility [fig_ref] Figure 6: The reproductive number [/fig_ref]. For example, the R 0 of wild-type SARS-CoV-2 is 1, which means one infected person transmits the virus to one person. The R 0 for the Alpha variant is 1.76, indicating more transmissibility [fig_ref] Figure 6: The reproductive number [/fig_ref]. For the Delta variant, the R 0 value is 5.01 [fig_ref] Figure 6: The reproductive number [/fig_ref] , indicating that one person infected with the Delta variant can spread the virus to up to five other people. This is a high increase of transmissibility compared to wild-type SARS-CoV-2. However, there are no reliable data on R 0 for Omicron [fig_ref] Figure 6: The reproductive number [/fig_ref]. Based on preliminary infection in South Africa, it is postulated to be higher than the Delta variant. ## J o u r n a l p r e -p r o o f Regarding the severity of COVID-19 caused by Omicron, the knowledge is insufficient to reach any conclusions. However, there is some information available. For example, the COVID-19 caused by Omicron has new symptoms not described before regarding other variants, such as fatigue, body aches and headache. Additionally to these symptoms, one patient also presented a fever and a very high pulse rate. Some patients have been found to be positive for Omicron infection but without symptoms. Some vaccinated people have become infected with the Omicron variant, indicating this variant can to some extent, break immunity acquired with vaccination. The escape from the immune system of vaccinated people might be related to the higher number of mutations in the RBD region, allowing the escape from anti-RBD neutralizing antibodies. It has been indicated but not confirmed that Omicron can escape from memory immunity conferred by T cells and durable immunity. Among vaccinated people infected with Omicron, some are asymptomatic and some have only mild symptoms. There have been no severe cases, hospitalization, or death caused by Omicron. This indicates that vaccination does not prevent Omicron infection, but it protects from severe cases, hospitalization, and death [bib_ref] Heavily mutated coronavirus variant puts scientists on alert, Callaway [/bib_ref]. This information suggests how important it is to expand vaccination worldwide to prevent severe COVID-19 caused by the Omicron variant. ## Interaction of s protein from variants with ace2 Although it is known that the S protein from variants has a higher affinity for ACE2 than the wild-type version, nobody has quantified this interaction. What is known and accepted is that variants are more transmissible than wild-type versions. Here, we have used the wild-type S protein from the Protein Data Bank (https://www.rcsb.org/, PDB ID: 6Z97) to build the three-J o u r n a l P r e -p r o o f dimensional (3D) models of protein from the Alpha, Beta, Gamma, Delta and Omicron variants using the Swiss Model Server (https://swissmodel.expasy.org/) [bib_ref] SWISS-MODEL: homology modelling of protein structures and complexes, Waterhouse [/bib_ref]. All the structure showed in the manuscript were validated. The models were created using the SWISS-model that already make the check in the structure using the Molprobity. Additionally, we have checked the structures on Molprobity server (http://molprobity.biochem.duke.edu/) [bib_ref] MolProbity: More and better reference data for improved all-atom structure validation, Williams [/bib_ref] by Ramachandran plot analysis. Also, the global quality factor and the reliability of the models' folding were evaluated by ERRAT2 (http://servicesn.mbi.ucla.edu/ERRAT/) and Verify3D (http://servicesn.mbi.ucla.edu/Verify3D/), respectively. The 3D model built was docked against the ACE2 (PDB ID: 7KMD) using the FRODOCK Interactive protein-protein docking server (http://frodock.chaconlab.org/) [bib_ref] FRODOCK 2.0: fast protein-protein docking server, Ramírez-Aportela [/bib_ref]. The FRODOCK server provides a docking score (DS) that indicates the interaction between the protein and ligand. In this case, higher DS values are associated with higher affinity of the interaction. Based on the corresponding DS value, this only enables suggesting which protein has more affinity than any other protein. As a control, the interaction between the wildtype S protein from Wuhan isolate with ACE2 [fig_ref] Figure 7: Docking analysis of Spike protein with ACE2 receptor [/fig_ref] has been used. This interaction has a DS value of 3025.00 [fig_ref] Figure 7: Docking analysis of Spike protein with ACE2 receptor [/fig_ref]. All S proteins from variants have higher DS values, indicating they have a higher affinity for ACE2 than wild-type S protein [fig_ref] Figure 7: Docking analysis of Spike protein with ACE2 receptor [/fig_ref]. This was expected, because, to some extent, all variants are more transmissible than wild-type SARS-CoV-2 from Wuhan. The S proteins from Beta and Gamma variants presented DS values, respectively, of 3102.00 and 3200.00, which mean increases of 2.5% and 5.85% in the DS interaction with ACE2 [fig_ref] Figure 7: Docking analysis of Spike protein with ACE2 receptor [/fig_ref] and D). This result suggests the S protein from those variants has a slight increase in the ability to interact with ACE2 compared to the wild-type versions of S protein. # Conclusion It is clear that the S protein from variants has been changing with two goals: 1) to improve the interaction with ACE2; and 2) to escape from the immune system. Spike amino acid substitutions, deletions, and insertions (Omicron) have a direct impact on how neutralizing antibodies interact with S protein from variants, leading to reinfection and even infection of fully vaccinated people. However, it looks like the mutations in the spike of variants are more driven to improve the interaction with the ACE. The fast interaction with ACE2 reduces time when SARS-CoV-2 is exposed to neutralizing antibodies. The results presented lead to the conclusion that the Omicron S protein is optimized to bind to the ACE2 receptor and escape from the Final approval of the manuscript and submission: Pedro F.N. Souza. [formula] immune [/formula] # Ethical approval Not applicable. ## Declaration of competing interests All authors declare no conflicts of interest. ## References [fig] [ 8 ]: Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations -SARS-CoV-2 coronavirus / nCoV-2019 Genomic Epidemiology -Virological, (n.d.). https://virological.org/t/preliminarygenomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-anovel-set-of-spike-mutations/563 (accessed January 22, 2021).[9] Severe Acute Respiratory Syndrome (SARS), (n.d.). https://www.who.int/healthtopics/severe-acute-respiratory-syndrome#tab=tab_1 (accessed January 21, 2021).[10] Middle East respiratory syndrome coronavirus (MERS-CoV), (n.d.). [/fig] [fig] Figure 1: Scheme model showing all the mutation on Spike protein from variants. In this model is possible to all the shared mutations among the variants and all the unique mutation of each variant. Created with www.BioRender.com. [/fig] [fig] Figure 2: Schematic representation of the SARS-CoV-2 particle and spike protein. (A) SARS-CoV-2 particle closuring the viral genome. (B) Spike protein consists of the S1 (holding the NDT and RBD regions) and S2 (holding the FP, HR1 and HR2 regions) units. (C) The two conformations assumed by Spike protein. The closed conformation with RBD inside the structure and the open conformation with RBD exposed to recognize ACE2 receptor. Created with www.BioRender.com. [/fig] [fig] Figure 3: Scheme presenting the mutations on variants and structural alignments of the variants compared to wildtype version Spike protein from SARS-CoV-2. (A) Comparison of Spike protein sequences from wildtype version of SARS-CoV-2 and mutations on Spike protein presenting in the variants D614, Alpha and Beta. (B) structural analysis of Spike protein from J o u r n a l P r e -p r o o f wildtype version of SARS-CoV-2, (C) Spike protein from Alpha variant and (D) Spike protein from Beta variant. RMSD analysis indicates changes in the structure. [/fig] [fig] Figure 4: Scheme presenting the mutations on variants and structural alignments of the variants compared to wildtype version Spike protein from SARS-CoV-2. (A) Comparison of Spike protein sequences from wildtype version of SARS-CoV-2 and mutations on Spike protein presenting in the variants Gamma and Delta. (B) structural analysis of Spike protein from wildtype version of SARS-CoV-2, (C) Spike protein from Gamma variant and (D) Spike protein from Delta variant. RMSD analysis indicates changes in the structure. [/fig] [fig] Figure 5: Structural alignment among Spike protein from Wildtype and variants of SARS-CoV-2. (A) structural analysis of Spike protein from wildtype version of SARS-CoV-2, (B) Spike protein from Delta variant and (C) Spike protein from Omicron variant. RMSD analysis indicates changes in the structure. [/fig] [fig] Figure 6: The reproductive number (R 0 ) wildtype and variants of SARS-CoV-2. The R 0 of the variants is higher than the wildtype version of SARS-CoV-2 suggesting the variants are more transmissible. [/fig] [fig] Figure 7: Docking analysis of Spike protein with ACE2 receptor. (A) Docking analysis of Spike protein of Spike protein from (A) wildtype version, (B) Alpha variant, (C) Beta variant, (D) Gamma variant, (E) Delta variant and (F) Omicron variant with the ACE2 receptor. DS is a docking score produced by FRODDOCK server and indicates strength of interaction between proteins. [/fig] [table] Table 1 Table 2: Data comparison of COVID-19 with other viral diseases Variants of SARS-CoV-2 J o u r n a l P r e -p r o o f [/table]

Ganzfeld Stimulation or Sleep Enhance Long Term Motor Memory Consolidation Compared to Normal Viewing in Saccadic Adaptation Paradigm Adaptation of saccade amplitude in response to intra-saccadic target displacement is a type of implicit motor learning which is required to compensate for physiological changes in saccade performance. Once established trials without intra-saccadic target displacement lead to de-adaptation or extinction, which has been attributed either to extra-retinal mechanisms of spatial constancy or to the influence of the stable visual surroundings. Therefore we investigated whether visual deprivation ("Ganzfeld"-stimulation or sleep) can partially maintain this motor learning compared to free viewing of the natural surroundings. Thirty-five healthy volunteers performed two adaptation blocks of 100 inward adaptation trialsinterspersed by an extinction blockwhich were followed by a two-hour break with or without visual deprivation (VD). Using additional adaptation and extinction blocks short and long (4 weeks) term memory of this implicit motor learning were tested. In the short term, motor memory tested immediately after free viewing was superior to adaptation performance after VD. In the long run, however, effects were opposite: motor memory and relearning of adaptation was superior in the VD conditions. This could imply independent mechanisms that underlie the short-term ability of retrieving learned saccadic gain and its long term consolidation. We suggest that subjects mainly rely on visual cues (i.e., retinal error) in the free viewing condition which makes them prone to changes of the visual stimulus in the extinction block. This indicates the role of a stable visual array for resetting adapted saccade amplitudes. In contrast, visual deprivation (GS and sleep), might train subjects to rely on extra-retinal cues, e.g., efference copy or prediction to remap their internal representations of saccade targets, thus leading to better consolidation of saccadic adaptation. # Introduction Saccadic adaptation is a form of implicit motor learning [bib_ref] The characteristics and neuronal substrate of saccadic eye movement plasticity, Hopp [/bib_ref] which is required to compensate for physiological changes in saccade performance due to age and growth [bib_ref] Age-related performance of human subjects on saccadic eye movement tasks, Munoz [/bib_ref] as well as for consequences of injury [bib_ref] Adaptive programming of phasic and tonic components in saccadic eye movements. Investigations..., Kommerell [/bib_ref]. Experimentally, reactive saccades can be adapted using the double-step paradigm introduced by McLaughlin by moving the target during saccade execution outward or inward which is not perceived by the participant due to saccadic suppression [bib_ref] Parametric adjustment in saccadic eye movements, Mclaughlin [/bib_ref]. De-adaptation or extinction can be induced by execution of saccades of the same type and vector but without intra-saccadic target displacement (extinction trials) [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref] [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref]. The most important measure for saccade accuracy is the relation between saccade amplitude and target step amplitude, usually referred to as "gain". In healthy participants, saccadic adaptation does not lead to an impairment in spatial orientation. Although the motor program for saccade execution has been modified, space is still perceived as stable despite saccade-induced retinal image shifts (spatial constancy). To maintain spatial constancy despite saccade-induce shifts of the retinal image, spatial representation of gaze target is updated ("remapped"). The posterior parietal cortex (PPC) plays an essential role for this remapping [bib_ref] The updating of the representation of visual space in parietal cortex by..., Duhamel [/bib_ref] [bib_ref] Gaze-centered updating of visual space in human parietal cortex, Medendorp [/bib_ref]. However, during saccadic adaptation process, neurons in the lateral intra-parietal sulcus (LIP) [bib_ref] The lateral intraparietal area codes the location of saccade targets and not..., Steenrod [/bib_ref] still encode the location of the visual saccade target, but not the adapted motor vector of the impending saccade. In addition the cerebellar structures play an important role in saccadic adaptation [bib_ref] Cerebellar activation related to saccadic inaccuracies, Liem [/bib_ref] [bib_ref] Effects of structural and functional cerebellar lesions on sensorimotor adaptation of saccades, Panouilleres [/bib_ref] [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref] [bib_ref] Saccade and vestibular ocular motor adaptation, Schubert [/bib_ref] and its consolidation [bib_ref] A role for the parietal cortex in sensorimotor adaptation of saccades, Panouilleres [/bib_ref]. While PPC is more involved in the control of voluntary saccades, the cerebellum plays important roles in saccadic adaptation [bib_ref] A role for the parietal cortex in sensorimotor adaptation of saccades, Panouilleres [/bib_ref] [bib_ref] Transcranial magnetic stimulation and motor plasticity in human lateral cerebellum: dual effect..., Panouilleres [/bib_ref]. Saccade adaptation can be stored. The course of adaptation is a good example of fast motor learning which takes place on the synaptic level. Subsequent testing without intra-saccadic target shift should exhibit similar results as at baseline-if there were no memories of this adaptation process. However, some residual adapted gain is preserved from the end of an adaptation session, referred to as "retention". Moreover, relearning of the same saccadic adaptation reconsolidates a previous memory trace: adaptation occurs faster which is referred to as "savings" [bib_ref] Interacting adaptive processes with different timescales underlie short-term motor learning, Smith [/bib_ref] [bib_ref] Distinct short-term and long-term adaptation to reduce saccade size in monkey, Robinson [/bib_ref] [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref]. Thus, different learning and memory processes can be obtained from saccadic adaptation over time. There is an ongoing debate on why modification of saccadic gain by a single course of adaptation-so-called short term saccadic adaptation (STSA) [bib_ref] The characteristics and neuronal substrate of saccadic eye movement plasticity, Hopp [/bib_ref] -can still be measured by reflexive saccade paradigms after weeks [bib_ref] Long-lasting modifications of saccadic eye movements following adaptation induced in the double-step..., Alahyane [/bib_ref] or even months [bib_ref] Long term retention of saccadic adaptation is induced by a dark environmental..., Wang [/bib_ref] as long as the same saccade type, vector and amplitude is tested in the same context. This long term saccadic adaptation (LTSA) [bib_ref] Distinct short-term and long-term adaptation to reduce saccade size in monkey, Robinson [/bib_ref] is a slow-learning process which becomes prominent if retrieval takes place two or more hours later which has been shown similarly for the hand motor domain [bib_ref] The acquisition of skilled motor performance: fast and slow experience-driven changes in..., Karni [/bib_ref]. A phase of rest or sleep following a learning phase helps to consolidate an acquired skill even by a short period of time [bib_ref] Sleep-dependent learning and memory consolidation, Walker [/bib_ref]. Consolidated skills are usually robust against interference [bib_ref] Multiple shifts in the representation of a motor sequence during the acquisition..., Korman [/bib_ref]. Models of LTSA has been defined for saccadic [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref] [bib_ref] Spontaneous recovery of motor memory during saccade adaptation, Ethier [/bib_ref] and other motor adaptation [bib_ref] Interacting adaptive processes with different timescales underlie short-term motor learning, Smith [/bib_ref] [bib_ref] Consolidation patterns of human motor memory, Criscimagna-Hemminger [/bib_ref] [bib_ref] Dual adaptation supports a parallel architecture of motor memory, Lee [/bib_ref]. Under natural viewing conditions visual cues in our surroundings with distinct stimulus properties and spatial relations among each other serve as contextual cues [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref] [bib_ref] Long term retention of saccadic adaptation is induced by a dark environmental..., Wang [/bib_ref]. Consequently eye movements in everyday life are likely to recalibrate the adapted saccadic amplitude. But this does not seem to be the case: there is no reset of the adapted saccadic gain but rather a decrease of adaptation over time [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref] [bib_ref] Long-lasting modifications of saccadic eye movements following adaptation induced in the double-step..., Alahyane [/bib_ref] [bib_ref] Long term retention of saccadic adaptation is induced by a dark environmental..., Wang [/bib_ref]. To elucidate the role of visual stimulation between adaptation learning sessions different forms of visual deprivation have been studied in animal and human studies: When monkeys were visually deprived by opaque goggles or kept in darkness some residual amount of adaptation has been found since the first saccades were not at the gain level reached at the beginning of the previous adaptation session. In awake humans STSA was observed in most of the subjects after a period of visual deprivation (15 min): eye closure or selfgenerated saccades did not affect the adapted gain in an outward adaptation task [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref]. However, long term visual deprivation effects on STSA or LTSA in humans have not been studied so far. Exclusion of visual targets during wakefulness can be achieved by several methods. Keeping the subject in darkness or using opaque goggles prevents visual stimulation but induces fatigue, i.e. a sleep setting. Sleep is a unique condition in which subjects also execute saccades without physically seeing visual stimuli [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref]. These rapid eye movements during sleep (REMs) lack of visual feedback and a stable visual surrounding for spatial reference. Hence, sleep can be regarded as a variant of visual deprivation. Recent studies provide evidence that there is neural activity related to visual processing during sleep, especially during dreams (see [bib_ref] The 'scanning hypothesis' of rapid eye movements during REM sleep: a review..., Arnulf [/bib_ref] for review). Furthermore sleep improves memory consolidation in motor and oculomotor tasks [bib_ref] The memory function of sleep, Diekelmann [/bib_ref] [bib_ref] Sleep is required for improving reaction times after training on a procedural..., Gais [/bib_ref]. Apart from eye closure visual deprivation can be induced by Ganzfeld stimulation (GS), i.e. a completely homogeneous visual field surrounding the participant induced by white goggles [bib_ref] Optische Untersuchungen am Ganzfeld: II. Zur Phaenomenologie des homogenen Ganzfelds, Metzger [/bib_ref]. In this study we therefore investigated the role of natural visual input on the consolidation of adapted saccadic gain. We wanted to know (1) whether free saccadic exploration of a stable natural visual surround-i.e. a constant egocentric visual space-re-adapts the saccadic system and (2) whether visual deprivation or sleep following saccade adaptation facilitates its consolidation. Therefore, we analysed learning curves by the sequence adaptation-extinction-adaptation before and after two hours spent in different visual conditions following adaptation. Measures of savings, i.e. facilitation of saccadic re-adaptation, were of particular interest. Deterioration of re-adaptation has not been described before and will be referred to as 'less memory savings'. Normal unrestricted viewing served as control condition for two visual deprivation paradigms (sleep and GS). We deprived subjects selectively from visuo-spatial orientation by Ganzfeld stimulation which provides a homogenous illuminative stimulation of the whole visual field, without any fixation cues. We examined long term effects by recording subjects four weeks later again. # Methods ## Subjects Thirty-five volunteers without any known neurological or psychiatric disease participated in a saccadic adaptation task with two blocks of experiments and a break in between. They were randomly assigned to 3 groups, differing in how the break between the saccadic adaptation trials was spent: In condition #1 participants were visually deprived using goggles with homogenous Ganzfeld stimulation (GS) during the break of two hours (N = 11, 4 males, 7 females; range 21-25 years; mean age 23 years) whereas in condition #2, 9 other participants (5 males, 4 females; range 19-30 years; mean age 23 years) were allowed to view unrestrictedly during the two hours' break. In condition #3, fifteen volunteers (6 males, 9 females; range 19-27 years; mean age 23 years) were allowed to sleep. All subjects were students of the University of Luebeck who were naïve about the study's purpose. Their vision was normal or corrected-to-normal at the time of the experiment. Participants' were not allowed to use alcohol or caffeine prior to the experiments. All participants gave written informed consent before starting the experiments corresponding to the declaration of Helsinki. The studies were approved by the Ethics Committee of the University of Luebeck. ## Experimental protocols Subjects performed the experimental sequence "adaptation-extinction-adaptation" twice, before and after the intervention, i.e. (i) two hours spent in one of the 2 different visual conditions "Ganzfeld stimulation" or "viewing", or (ii) before and after a whole night of sleep. Saccadic adaptation performance after the intervention was taken as a sign of motor memory consolidation modulated by the visual conditions. Ganzfeld stimulation/viewing condition. Experiments started between 10 a.m. and 1 p.m. Participants conducted two blocks of the experiment (see [fig_ref] Table 1: Study design [/fig_ref] with a two hours' break in between. The subjects were either visually deprived or allowed to look around. These 2 hours were spent in a quiet, normally illuminated room while listening to calm audio books. Visual deprivation was achieved by goggles with white coloured glasses which induced homogenous Ganzfeld stimulation (GS) [bib_ref] Optische Untersuchungen am Ganzfeld: II. Zur Phaenomenologie des homogenen Ganzfelds, Metzger [/bib_ref] without any contours that might enable the subjects to focus. Goggles were put on immediately at the end of the first block and were put off directly before the second part of the oculomotor experiment. Consequently visual input was reduced to a white shade without any edges that may recalibrate the saccadic positioning system. Subjects were asked to keep their eyes open during GS. Subjects in the viewing condition were not allowed to read or to pursue any other activities apart from normal viewing during the two hours' break. In a previous study we showed that eye movements during mental imagination of semantic contents induce visual scanning behaviour that is comparable to free viewing [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref]. In order to induce a "normal" rest situation we did not record eye movements during the two hours break. Long-term effects. After four weeks all participants of the daytime experiment were recorded once again with the same procedure, but switched visual conditions and adaptation side (cross-over design). Sleep condition. Subjects spent two consecutive nights in our sleep laboratory, the first one for habituation without and the second one (experiment night) with the saccadic adaptation block ("adaptation1-extinction-adaptation2") in the evening and in the following morning. In both nights, brain, muscle and ocular activity were measured by polysomnography. In the evening of the experiment night, participants got prepared for the polysomnographic recordings and subsequently performed the first part of the oculomotor experiment. At the end of the block of trials while sitting in the completely dark room of the laboratory, subjects were blindfolded and guided from the oculomotor lab to the sleeping laboratory in the room next door where they laid down to sleep. In the completely darkened sleep laboratory subjects had no visual cues during the night. In the morning while lying in bed again subjects were restricted from viewing and were guided blindfolded to the experiment in the oculomotor lab. ## Experimental setup In the saccade experiments, subjects sat at a distance of 1.40 m in front of the screen in an otherwise dark room. Head position was stabilized by a chin rest. Subjects were instructed to follow the laser point as quickly and accurately as possible. Saccade stimuli were generated by a red laser point (diameter, 0.1°; laser diode HL 11, LISA Lasersystem, Katlenburg-Lindau; laser intensity 9% of maximum output). The stimuli were back-projected on a translucent screen (Marata screen, BKE, Nörten-Hardenberg, Germany) and controlled by two galvo scanners (GSI Lumonics, Munich, Germany). Eye movements were recorded by a video-based eyetracking system (Eyelink-II, SR Research Ltd., Mississauga, Ontario, Canada) at 500 Hz sampling rate. In the sleep condition, electroencephalography (EEG), electromyography (EMG) and electrooculography (EOG) were recorded (polysomnography) using Ag/AgCl electrodes. EEG electrodes were placed at positions C3 and C4 of the international 10-20 system. The reference electrode was fixed on the nose. EMG was derived from two electrodes placed on the chin over the right and left mentalis muscle. EOG electrodes were fixed at classical supraorbital and infraorbital positions (for vertical eye movements) of one eye and in the outer angles of both eyes (for horizontal eye movements). A ground electrode was fixed on the forehead. Data were amplified (BrainAmp MRplus, Brain Products, Gilching, Germany) and recorded with a sampling rate of 500 Hz. EOG was DC-recorded, for EEG and EMG, a 250 Hz low-pass filter and a 0.015 Hz high-pass filter were set (time constant 10 s). Sleep was scored offline according to standard criteria. ## Experimental paradigms The oculomotor experiment consisted of several blocks of trials as shown exemplarily for one subject in [fig_ref] Fig 1: Example data from subject #4, blindfolded condition [/fig_ref] Saccade adaptation was induced by using a McLaughlin double step saccade adaptation paradigm [bib_ref] Parametric adjustment in saccadic eye movements, Mclaughlin [/bib_ref]. Adaptation blocks. A fixation point was presented in the centre of the screen for a duration of 1200 ±200 ms. Subsequently the target reappeared at 10°horizontal position for 80 ms. As soon as the eye started to move the target reappeared at the same position (non-adapted side) or was moved centripetally by an intra-saccadic step of 4°(inward adaptation). Online saccade detection was performed by three-fold criteria: 1) eye velocity of the current sample (N) must have increased by more than 30 deg/s since the previous sample (N-1) and 2) by more than 70 deg/s since the second last sample (N-2). 3) The eye position difference between sample N and N-3 had to be larger than1 deg. The lateral fixation point was presented for 1400-2200 ms and then smoothly moved back to the centre in a sinusoidal manner with a peak velocity of 15°/s in a time period of 500 ms. Smooth retrace of the target was used in order to exclude interference of centripetal saccade to centrifugal saccades. During adaptation blocks only one side was adapted which was randomly defined for each subject before the experiment started. Adaptation blocks consisted of 100 trials with 75 trials in the adapted direction and 25 trials into the non-adapted direction without intra-saccadic displacement. The order of the stimuli was pseudo-randomly intermixed (balancing trials) [bib_ref] Eye position effects in saccadic adaptation, Havermann [/bib_ref]. Baseline blocks. 40 trials with 10°target steps were performed, starting with central fixation for 1200 ms ±200 and subsequent peripheral stimulus presentation again for 1200 ms ±200. Extinction blocks. 50 trials (25 to the left and 25 to the right) were presented in the same way as described in baseline blocks. # Data analysis Horizontal and vertical eye positions were analyzed offline using Matlab (R2012a, The Mathworks Inc., Natick, MA, USA). Eye position data were calibrated and filtered (Gaussian filter, 100 Hz). Eye velocity was calculated as the difference of median eye position of four data points before and after the actual data point. Eye movements with an angular velocity >30°/s and an amplitude >0.3°were marked as saccades [bib_ref] Sleep is required for improving reaction times after training on a procedural..., Gais [/bib_ref] and checked interactively. Saccade amplitude was computed as the difference between eye positions at the end and at the start of a saccade. Saccade latency was computed as the time between the onset of the target shift and the onset of the saccade. Trials were excluded from further analyses because of blinks, anticipatory saccades (latency <70 ms) or extremely long reaction times (latency >800 ms) (3.0% ± 0.7 of the trials of the study in the sleep condition, 6.6% ± 2.4 in the visually deprived condition and 3.8% ±0.9 in the viewing condition). Saccadic gain was calculated as the ratio between saccade amplitude and amplitude of target displacement. In order to estimate the performance in baseline and extinction blocks a linear regression was computed (by robustfit function within Matlab). For the learning process of adaptation exponential fits of the saccade gain were performed using fminsearch function within Matlab which uses a Nelder-Mead downhill-simplex algorithm [bib_ref] Convergence Properties of the Nelder-Mead Simplex Method in Low Dimensions, Lagarias [/bib_ref]. From the exponential fit saccadic gain was derived after trial #1, 3, 5, 10, 15, 20 and 25 into the direction of adaptation. The regression curve at the beginning and the end of the block are referred to as 'initial offset' and 'final adaptation gain' hereafter. In order to quantify individual learning performance we calculated indices for memory retention of motor learning relative to baseline [bib_ref] Long-lasting modifications of saccadic eye movements following adaptation induced in the double-step..., Alahyane [/bib_ref] : Percentual gain changes were calculated for these different gains in adaptation and extinction blocks by the formula [formula] PGC n ¼ gain n À gain baseline gain baseline Ã100 [/formula] where n is the selected trial number (see above). The amount of retentionafter the night of sleep or the 2 hours' break respectively was calculated by the formula [formula] Retention n ð Þ ¼ PGC n ð Þ at postadaptationðmÞ PGC end ð Þ at adaptation2 Ã100 [/formula] where n indicates the trial number (1-99), m the sequence of postadaptations (1 or 2) and PGC (end) at adaptation 2 refers to the last trial before the (two hours or one night) break [fig_ref] Table 1: Study design [/fig_ref]. Statistical significance was assessed by using analyses of variance (ANOVAs) with the between-subject factor CONDITION (sleep, visual deprivation, viewing condition), the repeatedmeasures factor TIME for the different time-points of measures of PGC or Retention (at trial #1, 3, 5, 10, 15, 20, 25, 100) and BLOCK for blocks of trials (baseline, adaptation 1, extinction, adaptation 2, post-adaptation 1, post-extinction, post-adaptation 2). The factor SESSION depicts retests after four weeks of subjects that participated in daytime experiments (visual deprivation or viewing condition). Pairwise comparisons were performed by using post-hoc Scheffé tests and T-tests. All tests were performed using SPSS (version 21, IBM Inc. NY/USA). If not stated otherwise, subsequently reported values are means (±1 SE). # Results Eye amplitude adapts to final target position in adaptation trials in an exponential manner. [fig_ref] Fig 1: Example data from subject #4, blindfolded condition [/fig_ref] shows saccadic gains of a single subject in the experimental condition with visual deprivation. In all analyses the subjects adapted to the new target location; ANOVAs always showed a significant factor TIME with p always <0.001. The factor TIME is not of particular interest, therefore we will subsequently only report TIME x CONDITION interactions or differences between conditions. Sleep in the laboratory was normal (see [fig_ref] Table 2: Sleep results. [/fig_ref] with sleep durations between 427 and 548 minutes (mean ±standard deviation, 494 min ±35). The time needed to sleep onset varied between 6.5 and 36.5 minutes (mean ± standard deviation, 16.4 min ±9.8). Comparisons of average sleep stages in 1st (habituation night) and 2nd night (experimental night) revealed no significant differences (t-Test, p always !0.176). The percentage of the sleep stages did not correlate with PGC or retention (p always >0.64). ## Saccadic adaptation before intervention Initial saccadic gain during the baseline did not differ between conditions (0.95 (±0.02) in the sleep condition, 0.95 (±0.01) in the viewing condition, 0.95 (±0.02) in the visual deprivation condition; F(2, 32) = 0.022, p = 0.978). Learning progress was the same for all three conditions: ## First saccade after intervention After the intervention (factor CONDITION, i.e. free viewing, visual deprivation, sleep) PGC of the first saccade, i.e. comparison to baseline, failed to reach level of significance (F(2, 31) = 2.03, p = 0.148, [fig_ref] Fig 2: Percentual gain change of all adaptation blocks in relation to baseline gain [/fig_ref]. Retention value of the first saccade after intervention, i.e. comparison to adaptation right before intervention, showed significant effect of CONDITION (F(2, 31) = 4.1, p = 0.027, [fig_ref] Fig 3: Memory retention of the asymptotic adapted gain at the end of Adaptation... [/fig_ref]. Post-hoc multiple comparisons revealed a trend for differences between GS and free viewing (mean retention: 20.3% ±15.5, 73.5% ±18.9 respectively; p = 0.075) and significant differences between free viewing and sleep (mean retention sleep condition: 18.6% ±11.3; p = 0.041). GS and sleep condition did not differ (p = 0.997). Note that in the free viewing condition retention was more than three times higher as compared to GS and sleep condition. ## Re-learning after intervention: comparison with previously adapted gain During post-adaptation subjects rapidly readapted their saccade gain. The retention of previously learned motor adaptation after intervention did not show any TIME x CONDITION interactions, but CONDITIONs differed significantly (F(2, 31) = 5.28, p = 0.011). In postadaptation 1 subjects in the free viewing condition showed significantly more memory retention compared to those in the GS condition (p = 0.044) and the sleep condition (p = 0.003, [fig_ref] Fig 3: Memory retention of the asymptotic adapted gain at the end of Adaptation... [/fig_ref]. Post-hoc ANOVAs for each level of TIME revealed significant differences between conditions for up to 25 trials. Multiple comparisons showed that free viewing and sleep condition differed significantly (p always <0.039); a trend could be seen for comparisons of subjects in the free viewing condition and the GS condition for trials 3 and 5 (p <0.085). This means that the better memory retention of subjects in the viewing condition right after the intervention remains significant during the first 3 to 25 trials of the block post-adaptation 1. [fig_ref] Fig 2: Percentual gain change of all adaptation blocks in relation to baseline gain [/fig_ref]. Subtracting PGC post-adaptation 2 minus post-adaptation 1 reveals a memory loss of subjects in the viewing condition compared to the GS and sleep condition: an ANOVA of TIME and CONDITION revealed a significant effect for CONDITION (F (2, 31) = 10.4, p <0.001). Post-hoc ANOVAS for each level of TIME revealed a significant effect for CONDITION for trial 1 to 15 (F(2, 31) always >3.9, p always <0.031). These comparisons showed that subjects in the viewing condition needed 15 trials to regain similar adaptation performance as compared to the other conditions (p always <0.024, [fig_ref] Fig 2: Percentual gain change of all adaptation blocks in relation to baseline gain [/fig_ref]. [fig_ref] Fig 4: Baseline gain of session 1 [/fig_ref]. Baseline performance on the adapted side of session 1 of subjects in the VD condition (gain: 0.95 ±0.01) was significantly higher compared with the same side in session 2 (gain: 0.91 ±0.02; T(10) = 3.80, p = 0.003) and with the opposite side (gain: 0.91 ±0.01; T(10) = 3.04, p = 0.013, [fig_ref] Fig 4: Baseline gain of session 1 [/fig_ref]. Gain reduction of the non-adapted side after four weeks did not reach level of significance comparing both sides in session 2 (same side: p = 0.063; opposite side: p = 0.161) with baseline gain in session 1. For the viewing condition there were no significant gain changes (p always >0.47). ## Long-term effects Analyses of gain changes during saccade adaptation in session 2 revealed about the same results as in session 1 but parameters of interest did not reach level of significance (p always >0.1). # Discussion The aim of this study was to investigate the influence of natural viewing conditions compared to different forms of visual deprivation (VD) on motor memory consolidation. We hypothesized that free viewing following an implicit visuo-motor learning process, i.e. saccadic adaptation, may affect a person's capacity of retrieving the learned motor behaviour. Therefore, we compared saccadic performance in a paradigm with serial trials of an adaptation-extinction-adaptation sequence before and after two hours of normal viewing and VD, which was either presented by Ganzfeld stimulation (GS) or by a whole night of sleep. Consolidation of learned behaviour, i.e. adaptation retrieval, was assessed by the relearning performance (readaptation). The adapted saccadic gain was better preserved following free visual scanning in a natural environment indicating better short term motor memory than after VD. However, this motor memory turned out to be short lasting since the adapted saccadic gain could less be maintained for subjects in the viewing condition: after an additional extinction block Differential Effects of Visual Deprivation on Saccadic Adaptation saccadic adaptation performance decreased for subjects in the viewing condition whereas in both conditions of VD adaptation performance improved (savings). Hence, VD might contribute to a long term consolidation of the previously adapted saccade gain which was confirmed by the four week follow-up data. ## Impact of rest in normal viewing conditions on adapted saccadic gain A novel and striking result of our study is an increased storage of adapted gain after two hours of free viewing compared to visual deprivation. Saccadic activities during rest periods show differential effects on motor memory consolidation. Previous studies showed that visual input did not completely extinct adapted motor behaviour even on longer time scales [bib_ref] Long term retention of saccadic adaptation is induced by a dark environmental..., Wang [/bib_ref] [bib_ref] Characteristics of simian adaptation fields produced by behavioral changes in saccade size..., Noto [/bib_ref]. Extinction of adapted gain depended on the similarity of the learned adapted saccadic type and vector and the saccade type performed during the rest period [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref] [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref]. In our study subjects in all conditions were exposed to visual stimulation during rest which was completely different from the adaptation condition. In previous studies adapted saccade gain decreased stronger when reflexive saccades to targets without intrasaccadic displacement were performed during rest periods [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref] [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref]. Adapted gain was better maintained when self-induced or even no saccades were performed during rest. In a monkey study Seeberger et al. [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] compared the effects of reflexive saccades during rest with or without visible target after saccade onset and 20 minutes in darkness. Reflexive saccades with visible targets strongly reduced adapted gain whereas invisible targets trials nearly did not reduce adapted gain. These results seem to be in contrast to our study, but there are several differences that may account for it: Reflexive saccades grossly differ from voluntary saccades with or without visual feedback [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref]. That means (1) the conditions "reflexive saccade trials with permanent visual targets" and "free viewing", i.e. voluntary saccades with potential visual targets in the present study, are of different type. Additionally voluntary saccades without visual targets (GS) differ from reflexive saccades without visual feedback as used by Seeberger et al. [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] ; (2) the duration of rest was much longer in our study (2 hours, LTSA) as compared to the monkey study (20 min), i.e. STSA; and (3) the speed of saccade adaptation in monkeys differs from humans. For the rest period Gaveau et al. [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref] have demonstrated that only reflexive saccades of the same amplitude and direction as in the adaptation blocks have been effective to reduce adapted gain, but not self-generated saccades. In our free viewing condition, subjects performed mostly self-generated voluntary saccades, though visually-guided, but not primarily reflexive saccades. Further, voluntary saccades in free viewing are usually smaller than 5-7°, i.e. smaller than the target amplitude used in our adaptation paradigm [bib_ref] Visual Fixation Durations and Saccade Amplitudes: Shifting Relationship in a Variety of..., Pannasch [/bib_ref]. Thus our finding of a better storage of adapted saccade gain after free viewing (as compared to GS or sleep condition) can be attributed to the fact that saccades performed during free viewing were of totally different type and amplitude than those performed during the adaptation trials. Obviously, the spatial programming of adapted reflexive saccades was not influenced by the programming of voluntary saccades during free viewing. Consistent with this view a study on the execution of consecutive reflexive and voluntary saccades revealed that both types of saccades can be programmed in parallel, probably using a common motor map [bib_ref] The parallel programming of voluntary and reflexive saccades, Walker [/bib_ref]. Also functional imaging studies showed different cortical substrates activated during the execution of reflexive and voluntary saccades: during voluntary saccades blood oxygen level dependent (BOLD) response was greater in the lateral portions of the frontal eye fields (FEF), in the SEF and the left intraparietal sulcus [bib_ref] Differential cortical activation during voluntary and reflexive saccades in man, Mort [/bib_ref] [bib_ref] Activation of frontoparietal cortices during memorized triple-step sequences of saccadic eye movements:..., Heide [/bib_ref]. On the other hand reflexive saccades showed a greater BOLD response in the angular gyrus of the inferior parietal lobe, mostly in the right hemisphere. Contextual cues during rest may influence savings of saccadic adaptation, e.g. eye position [bib_ref] Eye position effects in saccadic adaptation, Havermann [/bib_ref] [bib_ref] Eye position specificity of saccadic adaptation, Alahyane [/bib_ref] , head position [bib_ref] Sensory, motor, and combined contexts for context-specific adaptation of saccade gain in..., Shelhamer [/bib_ref] , visual attributes of the target (flickering of the target [bib_ref] Saccade adaptation specific to visual context, Herman [/bib_ref] , target distance [bib_ref] Specificity of saccadic adaptation in three-dimensional space, Chaturvedi [/bib_ref] or darkness [bib_ref] Long term retention of saccadic adaptation is induced by a dark environmental..., Wang [/bib_ref]. Hence, saccadic gain can be reproduced and modified best in the experimental condition of learning, i.e. improved but also un-learned in a setting without changing these kind of features. In our study subjects in all conditions were exposed to visual stimulation during rest which was completely different from the adaptation (learning) condition. ## Visual conditions during rest To date, visual deprivation in saccade adaptation in humans has never been induced by GS or sleep. In non-human primate studies monkeys' were sitting in a dark room where they were able to sleep [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] or their heads were covered [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref]. In human studies participants were asked to close their eyes to achieve visual deprivation [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref]. Visual deprivation by eye closure comprises several components that discriminate this condition from free viewing: Apart from absent light stimuli eye movements are not accompanied by visual feedback implying that a visuo-spatial reference frame is missing. This may influence visuo-motor processing in different ways. Brain activity, e.g. differs between open and closed eyes in darkness [bib_ref] Eye closure in darkness animates sensory systems, Marx [/bib_ref]. Moreover saccadic dynamics differ with the eyes open and closed which has been related to saccade generation during eye closure [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref] [bib_ref] Differential effects of blinks on horizontal saccade and smooth pursuit initiation in..., Rambold [/bib_ref]. Accordingly, eye closure seems to affect saccade processing. Changes in luminance intensity also seem to affect saccade-related neural activation [bib_ref] Extraretinal saccadic signals in human LGN and early retinotopic cortex, Sylvester [/bib_ref]. Therefore we kept luminance intensity in our GS design stable. The use of GS as an instrument for visual deprivation allows selectively examining the role of fixation cues on consolidation of adapted saccadic gain. During sleep lids are usually closed but rapid eye movements (REM) do occur. The question arises as to whether REMs can be compared to voluntary eye movements without visual information. In sleep studies subjects reported processing visual scenes during sleep which they visually explored [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref] [bib_ref] The 'scanning hypothesis' of rapid eye movements during REM sleep: a review..., Arnulf [/bib_ref]. In a study on REM sleep disorder patients vividly processed their dreams [bib_ref] Do the eyes scan dream images during rapid eye movement sleep? Evidence..., Leclair-Visonneau [/bib_ref]. Likewise visual cortical regions show activation pattern during REM-sleep that resemble pontine-geniculate-occipital (PGO) waves in animals [bib_ref] fMRI evidence for multisensory recruitment associated with rapid eye movements during sleep, Hong [/bib_ref] [bib_ref] Human brain activity time-locked to rapid eye movements during REM sleep, Miyauchi [/bib_ref]. Saccade kinematics revealed that REMs are similar to voluntary eye movements without visual information whereas spontaneous eye movements in darkness were completely different from all other visual conditions [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref]. Thus, both, REM-sleep and GS provide about comparable conditions without visual feedback. ## Visual deprivation enables a stable long term consolidation The second main finding of our study is a stronger relearning of subjects in the VD condition (Ganzfeld stimulation or sleep): in the relearning phase-after a block of adaptation and extinction (post-adaptation 2)-participants of the free viewing condition showed less adapted gain whereas participants of both visually deprived conditions revealed a faster re-learning of adaptation. This indicates better long-term motor memory consolidation of subjects in the VD condition as compared to the free viewing condition. Four weeks later participants of the VD condition still showed remnants of gain adaptation which, however, was not specific to the adapted side. Previous studies indicated that visual deprivation may facilitate retrieval of adapted gain of saccades. In monkeys adapted saccade gain was partly maintained after one night sleep in a dark cage [bib_ref] Characteristics of saccadic gain adaptation in rhesus macaques, Straube [/bib_ref] or after 20 hours blindfoldedness [bib_ref] Distinct short-term and long-term adaptation to reduce saccade size in monkey, Robinson [/bib_ref]. As previously cited Seeberger et al. [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] studied saccade adaptation before and after blindfoldedness for 20 min. They reported a better maintenance of adapted saccade gain during blindfoldedness compared to extinction trials over this interval. In a related study relearning of saccade adaptation was faster after 30 min of blindfoldedness in a dark cage as compared to 30 min with extinction trials [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref]. In a study with human participants visual deprivation elicited a complete storage of adapted saccade gain [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref]. In contrast our data show partial maintenance of adapted gain in the first block after rest-but it was less than final adaptation gain before intervention. Our experimental design differed from this study: First, Gaveau et al. [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref] used an adaptation paradigm with a centrifugal intra-saccadic target displacement leading to an increased gain (gain up or outward adaptation), whereas in our study gain was decreased by a centripetal target displacement (gain down or inward adaptation). There is an ongoing debate as to whether up and down gain adaptation are related to different neural substrates [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref]. Zimmermann [bib_ref] The reference frames in saccade adaptation, Zimmermann [/bib_ref] found that gain up adaptation shows involves spatial target remapping, while gain down adaptation preferably engages motor learning. Therefore we assume that behavioural differences might be related to the different adaptation paradigms. Second, retention after 15 minutes of rest can be regarded as STSA whereas two hours belong to long-term consolidation (LTSA). Third, visual deprivation by sitting in the dark is different from GS or sleep. Fourth, we investigated complete learning curves of adaptation instead of the first trials only (e.g. [bib_ref] Self-generated saccades do not modify the gain of adapted reactive saccades, Gaveau [/bib_ref]. The slope of learning curves over 150 saccades was determined in only one non-human primate study [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref]. In line with previous reports our human participants adapted their saccade gain after about 50 trials, i.e. much shorter than in monkeys [bib_ref] The characteristics and neuronal substrate of saccadic eye movement plasticity, Hopp [/bib_ref]. We calculated the memory retrieval for several points on the fitting curve. These results are in line with monkey data from Kojima et al. [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref] : we showed significant differences between conditions in adaptation performance only in the beginning of the subsequent adaptation course after a 2 hours rest or sleep. Long term motor consolidation of saccadic adaptation. Following rest, the second adaptation block (post-adaptation 2, [fig_ref] Table 2: Sleep results. [/fig_ref] showed an inverse effect: adaptation gain was better maintained and better relearned compared to post-adaptation 1 in the VD conditions. Furthermore remnants of saccadic adaptation were still found after four weeks. Adapted gain achieved within one single course of adaptation is referred to as short term saccadic adaptation (STSA) [bib_ref] The characteristics and neuronal substrate of saccadic eye movement plasticity, Hopp [/bib_ref]. Adapted gain can still be detected 5 days after learning in humans. This may imply diverse learning processes at multiple timescales, which has been suggested in several models of saccadic adaptation [bib_ref] Memory of learning facilitates saccadic adaptation in the monkey, Kojima [/bib_ref] [bib_ref] Spontaneous recovery of motor memory during saccade adaptation, Ethier [/bib_ref] [bib_ref] The relative importance of retinal error and prediction in saccadic adaptation, Collins [/bib_ref] and other types of motor adaptation [bib_ref] Interacting adaptive processes with different timescales underlie short-term motor learning, Smith [/bib_ref] [bib_ref] Consolidation patterns of human motor memory, Criscimagna-Hemminger [/bib_ref] [bib_ref] Dual adaptation supports a parallel architecture of motor memory, Lee [/bib_ref]. Our short-and long-term results support motor learning of saccade adaptation at multiple timescales, as shown by the context-dependent (VD vs. free viewing) change of saccadic gain over time and exercise. A stable adapted gain based on consolidation of motor memory should be reflected in a high resistance against extinction. Collins and Wallman [bib_ref] The relative importance of retinal error and prediction in saccadic adaptation, Collins [/bib_ref] have demonstrated that prediction of the final target position has a stronger and more long-lasting influence on saccadic adaptation than the retinal error signal. In our experiment adapted gain was more vulnerable to extinction after free viewing than after visual deprivation or sleep. We suggest that subjects mainly rely on visual cues (i.e. retinal error) in the free viewing condition which could make them prone to changes of the visual stimulus in the extinction block. This indicates the role of a stable visual array for resetting adapted saccade amplitudes. In contrast, in our visual deprivation (GS and sleep), subjects are trained to rely on extra-retinal cues, e.g. efference copy or prediction to remap their internal representations of saccade targets. The smaller saccadic adaptation in the first post-adaptation block may be related to reference frame interference since probably because remapping of efference copy signals interfered with remapping of the target shift. In the second post-adaptation block visual cues and efference copy turned out to be unmatched. Therefore, saccadic adaptation following extinction appeared to be mainly influenced by prediction, which is regarded to be the most stable signal for the consolidation of motor memory with respect to the adapted saccade goal [bib_ref] The relative importance of retinal error and prediction in saccadic adaptation, Collins [/bib_ref]. After four weeks we found remnants of saccade adaptation which is in a line with a previous study [bib_ref] Long-lasting modifications of saccadic eye movements following adaptation induced in the double-step..., Alahyane [/bib_ref]. Given the known vector-specificity of saccadic adaptation we expected a uni-directional decrease in reflexive saccades but we found a bi-directional decrease. Obviously side specificity diminishes after a longer period of time and remaining gain modification is not related to the vector of the adaptation. We can exclude a non-specific decrease in saccade gain because subjects in the viewing condition did not show a similar decrease in saccade gain as compared to subjects in the GS or sleep condition. Neurophysiological correlates have been addressed previously: while prediction is generated presumably in cortical areas such as the prefrontal and anterior cingulate cortex, the pre-supplementary eye field and the hippocampus [bib_ref] Functional neuroanatomy of anticipatory behavior: dissociation between sensory-driven and memory-driven systems, Simo [/bib_ref] , a large number of studies has shown that more downstream cerebellar structures play a crucial role in saccadic adaptation processes [bib_ref] Cerebellar activation related to saccadic inaccuracies, Liem [/bib_ref] [bib_ref] Sensorimotor adaptation of saccadic eye movements, Pelisson [/bib_ref] [bib_ref] Saccade and vestibular ocular motor adaptation, Schubert [/bib_ref] [bib_ref] Effect of inactivation and disinhibition of the oculomotor vermis on saccade adaptation, Kojima [/bib_ref] which in turn correspond with the posterior parietal cortices for computing visuo-spatial constancy across saccades [bib_ref] Spatial constancy mechanisms in motor control, Medendorp [/bib_ref]. When there is no visual feedback following saccadic adaptation, such as during GS or REM sleep in our experiment, trans-saccadic visuo-spatial constancy cannot be maintained sufficiently by visual cues or efference copy, we assume that the cerebellar processing of adaptation is more and more controlled by these prefrontal areas involved in prediction, thereby supporting consolidation of motor memory. This effect may be masked at first by predominant parietal functions of remappping. Sleep does not boost motor memory consolidation in saccadic adaptation. Many motor skills benefit from sleep as their memory consolidation occurs during sleep [bib_ref] The memory function of sleep, Diekelmann [/bib_ref]. This made us suggest that that retention of adapted saccadic gain might benefit from sleep as well. In two studies monkeys were blindfolded for at least one night and re-adapted the next day [bib_ref] Distinct short-term and long-term adaptation to reduce saccade size in monkey, Robinson [/bib_ref] [bib_ref] Characteristics of saccadic gain adaptation in rhesus macaques, Straube [/bib_ref] , i.e. identical visual deprivation as compared to our sleep condition. Both monkey studies report a large variation of retention on the next day, ranging from no washout to complete washout with an average in-between. Our data are in line with these results. Unfortunately in both studies there were no direct comparisons with free viewing conditions. This comparison can only be deduced from monkey data [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] in which 15 minutes of visual deprivation was applied, where the monkeys showed signs of light sleep. Right after, there was nearly no washout of adapted gain which stands in contrast to related monkey studies [bib_ref] Distinct short-term and long-term adaptation to reduce saccade size in monkey, Robinson [/bib_ref] [bib_ref] Characteristics of saccadic gain adaptation in rhesus macaques, Straube [/bib_ref] and to our data. We assume that this might be due to the short duration of sleep and possibly to different sleep stages. To date there is no study which systematically varied saccadic adaptation washout related to the duration of sleep. In our data we did not find any differences in savings between visual deprivation during wakefulness (GS) and sleep. This might be related to the fact that sleep related memory consolidation holds for explicit motor tasks [bib_ref] Current concepts in procedural consolidation, Robertson [/bib_ref] [bib_ref] Sleep does not benefit probabilistic motor sequence learning, Song [/bib_ref]. In contrast, saccadic adaptation is an implicit form of motor learning [bib_ref] The characteristics and neuronal substrate of saccadic eye movement plasticity, Hopp [/bib_ref]. Rapid eye movements during sleep might have had an impact on motor adaptation but this was not the case: REM sleep duration was about two hours which is the same amount of time as the GS stimulation duration. Additionally REM sleep duration did not correlate with PGC. Hobson and Friston [bib_ref] Waking and dreaming consciousness: neurobiological and functional considerations, Hobson [/bib_ref] assume that there are processes of optimization of internal models during REM-sleep that rely on other feedback mechanisms than visual ones, such as proprioception. Indeed, in our experiment visual error was induced visually and hence not of a permanent systemic origin as it may be caused by muscle damage. It has been demonstrated that proprioception of the eye muscles affects vision [bib_ref] Eye proprioception used for visual localization only if in conflict with the..., Balslev [/bib_ref] [bib_ref] Influence of extraocular muscle proprioception on vision, Buisseret [/bib_ref] and probably also saccadic adaptation at longer timescales [bib_ref] The saccadic system, Leigh [/bib_ref]. It is thus possible that sleep contributes to recalibrate the STSA when visual feedback is not available. Furthermore, in previous studies we could show that sleep improves reaction time of saccades but not the gain [bib_ref] Sleep is required for improving reaction times after training on a procedural..., Gais [/bib_ref] [bib_ref] Differential effects of sleep deprivation on saccadic eye movements, Zils [/bib_ref]. In both studies saccadic gain of all participants was almost perfect right from the beginning, i.e. a ceiling effect might have suppressed sleep related motor learning. Now we can show that sleep does not boost motor memory consolidation in saccadic adaptation-at least compared to two hours of visual deprivation in wakefulness. Adapted gain after two hours of free viewing and visual deprivation: a consequence of non-disturbed spatial remapping?. Saccadic gain directly following the rest period was significantly higher after free viewing as compared to VD. In the absence of visual cues in the Ganzfeld or sleep condition, subjects may move their eyes to internally represented target positions [bib_ref] Eye movements during REM sleep and imagination of visual scenes, Sprenger [/bib_ref]. As there is no visual reference frame, remapping of space across each saccade requires updating of target positions by the use of extra-retinal information, i.e. efference copy or prediction. This procedure is neuronally computed mainly by the parietal eye field in the posterior parietal cortex [bib_ref] The lateral intraparietal area codes the location of saccade targets and not..., Steenrod [/bib_ref] [bib_ref] Activation of frontoparietal cortices during memorized triple-step sequences of saccadic eye movements:..., Heide [/bib_ref] , and does probably interfere with the remapping of the adapted eye position during the rest period. This could explain why subjects in the GS or sleep condition needed some 50 adaptation trials in the first block after the rest period to regain previous adaptation, at a time, when obviously the influence of prediction was not yet strong enough. The situation in the free viewing condition is different: There is no interference in terms of remapping, as free viewing provides a stable visual reference frame for maintaining spatial constancy across saccades on the basis of visual cues, not requiring the use of extra-retinal cues such as efference copy. Thus we suggest that spatial remapping interferes with memory retention of saccade adaptation in the first block after VD rest period. # Conclusions Our data show different effects of visual deprivation and free viewing on a precisely quantifiable motor learning, i.e. saccade adaptation. Adapted saccade gain is best consolidated in a natural viewing condition-if retrieved directly after intervention. In contrast, GS and sleep seem to support long-term consolidation of saccadic adaptation. To some extent our data are contradictory to a previous study [bib_ref] Non-visual information does not drive saccade gain adaptation in monkeys, Seeberger [/bib_ref] but several differences in the study design may have contributed to. Further research is needed to disentangle the influence of visual information during rest. From our data we speculate that voluntary saccades during free viewing are performed towards stationary visual targets within a stable visual reference frame. This setting does not interfere with the adaptation-induced remapping of visual reflexive saccades during the adaptation trials. In contrast, visual deprivation by Ganzfeld stimulation or sleep does not seem to rely on visual cues (retinal error) and thereby facilitates long term motor memory. It obviously makes adapted gain less vulnerable against extinction, even after four weeks of normal viewing, most probably by using extra-retinal cues for the position of the adapted saccade goal such as prediction. We suggest that VD may play a beneficial role in consolidation of saccadic adaptation as a motor learning program. [fig] Fig 1: Example data from subject #4, blindfolded condition. Eye position is plotted against trial number, dots symbolise saccadic amplitude. Linear fit was calculated for amplitudes of the non-adapted side (black and gray curves) and exponential fit for the adapted side (light and dark blue curves). Upper part shows data before 2 h of visual deprivation, lower part data of recordings afterwardsdoi:10.1371/journal.pone.0123831.g001 Differential Effects of Visual Deprivation on Saccadic Adaptation PLOS ONE | DOI:10.1371/journal.pone.0123831 April 13, 2015 [/fig] [fig] Fig 2: Percentual gain change of all adaptation blocks in relation to baseline gain. In adaptation 1 (A) and 2 (B) subjects show a similar improvement of learning performance in all conditions. After the 2h break/one night of sleep subjects in the free viewing condition show a significantly higher gain reduction than those in the VD condition (C, trial 3 to 10) which diminishes after a short extinction (D). Percentual gain change (E) plotted as the difference between Post-adaptation 2 minus Post-adaptation 1. In the VD conditions adaptation gain was further improved in the second part of the experiment while in the free viewing condition subjects showed a significant loss. Stars indicates significant effects for CONDITION in univariate ANOVAs for each trial depicted ('*' = p <0.05, '**' = p <0.01).doi:10.1371/journal.pone.0123831.g002 Differential Effects of Visual Deprivation on Saccadic Adaptation PLOS ONE | DOI:10.1371/journal.pone.0123831 April 13, 2015 At the end of post-adaptation 1 and 2 subjects in all conditions reached about the same level of adaptation, hence CONDITION did not differ (post-adaptation 1: [/fig] [fig] Fig 3: Memory retention of the asymptotic adapted gain at the end of Adaptation 2 achieved in Postadaptation 1 (A) and 2 (B). In the free viewing condition adapted gain was significantly better memorized compared to the sleep condition and a trend for GS condition. Stars indicate significant effects for CONDITION in univariate ANOVAs for each trial depicted ('*' = p <0.05, '**' = p <0.01). doi:10.1371/journal.pone.0123831.g003 [/fig] [fig] Fig 4: Baseline gain of session 1 (first recording) and session 2 (four weeks later) (mean ±SE). Data are shown for each side separately, i.e. hatched bars belong to the non-adapted side, plain bars to data of the adapted side. Stars indicate significant differences revealed by Student's T-Test ('*' = p <0.05, '**' = p <0.01). doi:10.1371/journal.pone.0123831.g004 [/fig] [table] Table 1: Study design. Condition A Baseline Adaptation 1 Extinction Adaptation 2 2 h visual deprivation Post-adaptation 1 Post-extinction Post-adaptation 2 [/table] [table] Table 2: Sleep results. [/table]

Which urban land covers/uses are associated with residents’ mortality? A cross-sectional, ecological, pan-European study of 233 cities Strengths and limitations of this study ► This was a large international study of 233 cities within 24 countries. ► We used high-quality, internationally comparable land cover/use data from the European Urban Atlas. ► We were able to examine the impact of land covers/ uses both collectively (via landscape metrics) and individually. ► Previously validated city-level standardised mortality ratio (SMR) data were spatially joined with contemporaneous land cover/use data. ► Although associations between land covers/uses and SMRs were described, we have no information on how the individuals used or were exposed to the land covers/uses.AbStrACtObjectives The study aim was to determine whether the range and distribution of all, and proportions of specific, land covers/uses within European cities are associated with city-specific mortality rates.Setting 233 European cities within 24 countries.Participants Aggregated city-level all-cause and agegroup standardised mortality ratio for males and females separately and Western or Eastern European Region. results The proportion of specific land covers/uses within a city was related to mortality, displaying differences by macroregion and sex. The land covers/uses associated with lower standardised mortality ratio (SMR) for both Western and Eastern European cities were those characterised by 'natural' green space, such as forests and semi-natural areas (Western Female coefficient: −18.3, 95% CI −29.8 to −6.9). Dense housing was related to a higher SMR, most prominently in Western European cities (Western Female coefficient: 17.4, 95% CI 9.6 to 25.2). Conclusions There is pressure to build on urban natural spaces, both for economic gain and because compact cities are regarded as more sustainable, yet here we offer evidence that doing so may detract from residents' health.Our study suggests that urban planners and developers need to regard retaining more wild and unstructured green space as important for healthy city systems. Strengths and limitations of this study ► This was a large international study of 233 cities within 24 countries. ► We used high-quality, internationally comparable land cover/use data from the European Urban Atlas. ► We were able to examine the impact of land covers/ uses both collectively (via landscape metrics) and individually. ► Previously validated city-level standardised mortality ratio (SMR) data were spatially joined with contemporaneous land cover/use data. ► Although associations between land covers/uses and SMRs were described, we have no information on how the individuals used or were exposed to the land covers/uses. # Abstract Objectives The study aim was to determine whether the range and distribution of all, and proportions of specific, land covers/uses within European cities are associated with city-specific mortality rates. Setting 233 European cities within 24 countries. Participants Aggregated city-level all-cause and agegroup standardised mortality ratio for males and females separately and Western or Eastern European Region. results The proportion of specific land covers/uses within a city was related to mortality, displaying differences by macroregion and sex. The land covers/uses associated with lower standardised mortality ratio (SMR) for both Western and Eastern European cities were those characterised by 'natural' green space, such as forests and semi-natural areas (Western Female coefficient: −18.3, 95% CI −29.8 to −6.9). Dense housing was related to a higher SMR, most prominently in Western European cities (Western Female coefficient: 17.4, 95% CI 9.6 to 25.2). Conclusions There is pressure to build on urban natural spaces, both for economic gain and because compact cities are regarded as more sustainable, yet here we offer evidence that doing so may detract from residents' health. Our study suggests that urban planners and developers need to regard retaining more wild and unstructured green space as important for healthy city systems. bACkgrOund In 2014, 54% of the world's population lived in urban environments; this is set to rise to 66% by 2050. 1 It is therefore important that urban environments are designed to enable and promote good health. Urban areas are complex systems and one emergent feature is the health of their residents. 2 There are stark between-city inequalities in population health [bib_ref] Multi-scalar influences on mortality change over time in 274 European cities, Richardson [/bib_ref] and previous work on trends and inequalities between European cities in sexspecific mortality rates suggests that a city environment itself is an important influence. [bib_ref] Sustainable urban forms: their typologies, models, and concepts, Jabareen [/bib_ref] [bib_ref] Socioeconomic inequalities in cause-specific mortality in 15 European cities, Marí-Dell&apos;olmo [/bib_ref] [bib_ref] Socioeconomic inequalities in injury mortality in small areas of 15 European cities, Gotsens [/bib_ref] The physical extent, design and composition of cities are important components of the urban system and are associated with the health and well-being of residents. [bib_ref] Role of built environments in physical activity, obesity, and cardiovascular disease, Sallis [/bib_ref] The diverse development trajectories of cities means there is considerable variety in their size, configuration and content.This variety provides a rich natural laboratory to study the implications for health of their design and construction. Given that most cities are subject to some kind of planning control and vision, there is also great potential for epidemiological insights into how the urban landscape affects health to impact on the lived experiences of billions of people. This study is about 'what is on the ground' in a city, which we refer to as land cover/uses. Land cover refers to what the surface actually is, for example, forests, concrete or water. Land uses are the purposes for which humans use the land, such as airports or roads. Urban land cover/use can influence health via multiple mechanisms including the promotion or inhibiting of healthy behaviours, [bib_ref] City planning and population health: a global challenge, Giles-Corti [/bib_ref] increasing exposure to pathogens such as air pollutants and noise 12 or offering psychologically restorative spaces. [bib_ref] Environmental, health, wellbeing, social and equity effects of urban green space interventions:..., Hunter [/bib_ref] Evidence suggests that land cover/ use exerts an independent influence on Open access residents, over and above residents' own characteristics. For example, the density of settlement and transport networks have been associated with variation in mortality risk across a number of English cities. 14 In a previous multicity European study, we found that the presence and quantity of specific land covers/uses within a city was associated with residents' reported life satisfaction. We also found that specific land covers/uses, and a more even distribution of the land covers/uses (as opposed to dominance by one or two land covers/uses), were both associated with lower levels of socioeconomic inequality in life satisfaction within the city. [bib_ref] Are urban landscapes associated with reported life satisfaction and inequalities in life..., Olsen [/bib_ref] We hypothesised that the range and distribution of land covers/uses speaks to the range of opportunities and environments, and hence affordances [bib_ref] Urban green space for health and well-being: developing an 'affordances' framework for..., Lennon [/bib_ref] that the city provides-and that this might explain the associations with life satisfaction levels and its equality. However, life satisfaction is fundamentally a subjective measure of well-being, and may be particularly sensitive to the environment. Therefore, in this study we focused on mortality which is, arguably, a more robust measure of population health. In doing so, we were able to include a substantially larger sample of European cities (n=233). No previous study has assessed associations between city-level land covers/uses and mortality in such a large and diverse international sample of cities. The study aim was to determine whether the range and distribution of all, and proportions of specific, land covers/uses within European cities is associated with the city-specific mortality rate. Previous research found that a city's wider macroregion (ie, its location in Eastern or Western Europe) was an important determinant of mortality rate, and moderator of association between environment and health, therefore this was also explored. Our specific research questions were: i. Is the range and distribution of land covers/uses within a city associated with city-level mortality rates? ii. Which, if any, specific land covers/uses are associated with city-level mortality rates? iii. How do these between associations vary with sex and by macroregion? # Methods ## Setting and data sources european urban atlas The 2006 Urban Atlas provided pan-European comparable land cover/use data collected and digitised from satellite data for urban areas.We selected data for this period to match the time period for the available mortality data. The Atlas distinguishes 26 different land cover/use categories at a 10 m 2 resolution. [fig_ref] Figure 1: Urban Atlas land use/cover categories and satellite base map [/fig_ref] provides an illustration of these data for part of one city. In this example, 20 different land uses are shown and labelled. Full land use descriptions are available in online supplementary table 1. The land cover/use data were matched to the same city administrative boundaries as the mortality data. geoprocessing of urban Atlas data We calculated the total area (km 2 ) for each land cover/ use by city, using an extension for ArcMap GIS called Patch Analyst.This was then converted to proportion of land cover/use by city (some values equalled zero if the land cover/use was not present in a city, for example, not all cities contained an airport). Following our previous work, [bib_ref] Are urban landscapes associated with reported life satisfaction and inequalities in life..., Olsen [/bib_ref] we then calculated landscape metrics. These are quantitative summaries of the range and statistical distribution of the various land covers/uses within a whole city. We calculated Shannon's diversity index (SDI) and Shannon's Evenness index (SEI)to assess the variety of all land covers/uses, and their relative availability, within each city. Only land covers/uses present within each city were included, this provided a relative measure of the diversity of land covers/uses available at the city level (SDI) and the distribution of area among the land covers/uses present within a city (SEI). No information was available about mortality rates either for socioeconomic groups or spatial locations within the city. We could not, therefore, assess within-city inequalities in this study. The SMR, land cover/use and GDP data were matched at city level. The final dataset included 233 cities (within 24 countries). The cities were allocated to either Western or Eastern-Central bloc macroregion, following Richardson et al, [bib_ref] Multi-scalar influences on mortality change over time in 274 European cities, Richardson [/bib_ref] referred to hereafter as Western or Eastern. There were 85 cities (in 11 countries) classed as Eastern. ## Gross domestic product ## Patient and public involvement No patients were involved in the development of the research question, design and implementation of the study or interpretation of the results. # Statistical analysis First, linear regression models assessed associations between the landscape metrics and city SMR data, adjusting for city GDP. Next, models for specific land covers/uses were built. Preliminary analyses suggested that associations between the specific land cover/use measures and SMR were not linear, so the former were categorised. To do this, the proportion of the total area covered by each land measure was calculated within each city, and then classified into quintiles. This process was carried out separately for each macroregion. We categorised the data into quintiles rather than apply polynomial terms in order to aid interpretation and therefore help guide policymakers. [bib_ref] A new approach to categorising continuous variables in prediction models: proposal and..., Barrio [/bib_ref] Furthermore, this approach also allowed us to generate the marginal estimates, which were plotted. These quintiles were then fit as factors, along with city GDP, in a linear regression, while also allowing for clustering within country. Each land measure was assessed separately using Wald statistics and subjected to post hoc pairwise comparisons using Sidak's correction for multiple testing across all terms.We chose not to include all land covers/uses in a singular model due to concern of multicollinearity, which was confirmed when tested (Eastern European Cities-VIF: 17.20, Western European Cities-VIF: 6.41). All analyses were run separately by sex and macroregion. Stata (SE V.14) [bib_ref] Stata statistical software: release 14, Statacorp [/bib_ref] and R (V.3.6.0)were used for analysis, with significance levels set at 5%. # Results For the models looking at specific land covers/uses we present our results in three ways. First, we highlight the land covers/uses for which we found strongest evidence of association with either lower or higher mortality rates [fig_ref] Table 1: Land covers/uses with strongest and most consistent evidence of association with city... [/fig_ref]. This selection was systematic, based on Wald test values and on both size and apparent 'dose response' of associations between quintiles of land cover/use and mortality. [fig_ref] Table 1: Land covers/uses with strongest and most consistent evidence of association with city... [/fig_ref] crudely 'ranks' these selected land covers/uses according to the extent to which they were associated with mortality in both macroregions, and both sexes (which we call 'universal'). A downward pointing arrow denotes that more of that land cover/use was associated with lower mortality, and an upward arrow that it was associated with higher mortality. Second, we present regression coefficients for these selected land covers/ uses ( . These figures convert the associations to estimated SMRs, perhaps a more meaningful expression of the results. The reader is invited to pay attention to the position on the Y axis, and slope, of the lines in the figures. We found no meaningful associations between SMRs and the landscape metrics and therefore do not report those results. land covers/uses associated with lower mortality In general, cities with a higher proportion of agricultural, semi-natural areas and wetlands enjoyed lower mortality rates (figure 2). In Eastern cities, this advantage weakened in cities with the highest proportion of this land use (figure 2, table 2). [fig_ref] Figure 2: Estimated standardised mortality ratios [/fig_ref] shows a somewhat large difference in SMR between both sexes for Western cities in quintile 1 and quintile 5. A higher proportion of forests within a city was also associated with lower SMR for females in both Eastern and Western European cities, but not males [fig_ref] Table 2: Regression coefficients of land covers/uses found to contribute to a model for... [/fig_ref]. In Western cities, the proportion of isolated structures (single dwellings surrounded on all sides with natural green land) was associated with lower SMRs for males, but in Eastern cities this protective association was present for females only [fig_ref] Table 2: Regression coefficients of land covers/uses found to contribute to a model for... [/fig_ref]. For Eastern European cities only, higher proportion of land containing sports and leisure facilities was associated with lower SMRs, although only significantly for men [fig_ref] Table 2: Regression coefficients of land covers/uses found to contribute to a model for... [/fig_ref]. land covers/uses associated with higher mortality The two land covers/uses most consistently associated with higher mortality were industrial, commercial, public and military, and green urban areas (figure 3). In Western European cities, higher proportions of the former were associated with higher SMRs, with a sharpening of association in cities containing the most (quintile 5). The association was not consistent or significant for men living in Eastern cities but was for women, although the trend fluctuated and only cities with greatest proportions of this land cover/use were associated with a higher SMR. A positive association between green urban areas and SMR was seen for Western women, and both sexes in Eastern cities (table 2, [fig_ref] Figure 3: Comparisons between standardised mortality ratio [/fig_ref]. The trend in Eastern cities suggested a threshold effect that may be substantially more important for health, with modest reductions in SMR as proportions increase from quintiles 1 to 4, and then a sudden and steep adverse association at quintile 5. More derelict land (land without current use) was associated with higher SMRs in the Eastern cities, with the greatest adverse association quintiles 4 and 5. Western European cities with higher proportions of land classified as residential and discontinuous low-density urban fabric had higher SMRs, particularly for cities in quintile 5 [fig_ref] Table 2: Regression coefficients of land covers/uses found to contribute to a model for... [/fig_ref] , suggesting a critical level at which these land covers/uses become substantially more important to health. diSCuSSiOn This is the first pan-European study to examine whether, and which, land covers/uses within a whole city are associated with mortality rates. By integrating health and environmental data from a large number of cities across Western and Eastern Europe, we found that the proportion of some specific land covers/uses within a city was related to mortality; most land covers/uses were not clearly associated with mortality and there were differences by macroregion and sex in association. We found no evidence that the overall distribution and balance of multiple land covers/uses, measured by landscape metrics, was related to mortality rates. Overall, we observed that higher proportions of natural spaces and less dense or non-residential land cover/use was associated with lower mortality. For example, greater proportions of lower density settlement (such as isolated structures) showed some association with lower SMR, while SMRs were higher for Western European cities with a greater residential proportion and low-density discontinuous cover (which is principally housing). Specifically, our most consistent finding was that presence of more 'relatively wild' green spaces, such as agricultural, seminatural areas and wetlands and forests was associated with lower SMRs, an association observed across sexes and macroregions and relatively strongly. The health benefits of contact with nature are very well established from both experimental and observational studies. [bib_ref] Nature and health, Hartig [/bib_ref] These kinds of spaces may be particularly restorative or conducive of leisure and recreation. Studies have shown the protective effects of natural environments and association with reduced risk of mortality. For example, two large Canadian studies examined green space using normalised difference vegetation index (NDVI) around residents' home and showed a higher NDVI was associated with lower mortality rates. Our results for the more natural spaces are in stark contrast to those for green urban areas, the proportion of which was relatively consistently associated with higher mortality. At first, this seems like a directly contradictory finding, but it is important to be clear what kinds of spaces are classified as 'green urban areas' in the Urban Atlas. Large urban parks are not classified solely as 'green urban areas' and will be separated into their designated components, for example, if a park contains a botanical garden, zoo or national trust gardens, these areas will be classified as an industrial or commercial areas; blue spaces within parks will be defined as waters; any space which can be used for sport or containing sport facilities (goal posts, cricket or basketball markings) will be defined as sports or leisure facilities. In this land use classification, 'green urban areas' are relatively small and manicured 'in-fill' green spaces within residential and commercial developments. Their association with greater mortality is not unprecedented in the literature. Previous work found that the total amount of green space within American cities was associated with higher mortality rates, suggesting that the indicator was capturing sprawling cities, however this study was unable to distinguish between the green space type. [bib_ref] Green cities and health: a question of scale?, Richardson [/bib_ref] In light of our findings, the American study may have been capturing the development of semi-natural areas into 'in-fill' green developments. We have previously found that greater amounts of 'green urban areas' in European cities were associated with reduced reported life satisfaction for its city residents. [bib_ref] Are urban landscapes associated with reported life satisfaction and inequalities in life..., Olsen [/bib_ref] The salutogenic effects of nature may be maximised by more natural areas and/or the 'pockets of green' in and among urban developments may be insufficient to offset the adversity of living in dense residential and commercial environments. There were some differences between region and sex in the direction of associations. We found that less desirable land types/uses within cities, such as land without use, were associated with higher SMRs for those living in Eastern European cities. Cities depend on their residents for economic, social, cultural and environmental prosperity and maintaining a diverse, skilled and satisfied residential population is vital for a city since their disenchantment could trigger a vicious downward spiral. [bib_ref] Branding the City as an attractive place to live, Insch [/bib_ref] Dereliction of cities has been linked to decreasing employment rates due to many individuals, particularly younger and educated, migrating from these areas to more prosperous cities [bib_ref] Environmental improvements in relation to derelict land, Ward [/bib_ref] and individuals living in areas with a high proportion of brownfield land are significantly more likely to suffer from poorer health than those with a lower proportion. [bib_ref] Healthy land? An examination of the area-level association between brownfield land and..., Bambra [/bib_ref] Although derelict and vacant land covers/uses remain in both Western and Eastern European regions and was a national problem in many Western cities during the mid/late 20th century as many industries declined or closed, [bib_ref] Environmental improvements in relation to derelict land, Ward [/bib_ref] there is country-specific evidence that areas of these land covers/uses are decreasing. For example, in Scotland from 2017 to 2018 derelict land decreased by 6% nationally,which may reduce the importance for population health. Perhaps the approaches taken by some Western governments to tackle it and spearhead regeneration in these areas, for example, have mitigated impacts. Furthermore, the impact of industrial, commercial, public and military spaces within a city seemed benign for men living in Eastern European Cities but not for women, and both sexes in Western European cities. Males and females in eastern cities also seemed to benefit from greater proportions of sports and leisure facilities. The western/eastern differences may reflect levels of city and economic development not adequately captured by our control for GDP. Perhaps, for example, a city in the east that has substantial amounts of land dedicated to sports and leisure facilities hosts a population with sufficient affluence to sustain them. Overall, our results present a challenge to healthy urban planning. Building on natural green spaces can address housing shortages, increase the local taxation base and support the development of local infrastructure (school, transportation, etc). Residential settlements with 'green views' command a premium price. Furthermore, a central message of contemporary urban planning is that dense and/or compact cities are 'sustainable'. Yet, the literature already hints that compact cities Open access characterised by high-density residential areas have both benefits and disadvantages for their residents. The environmental benefits, owing to the reduced carbon emissions required in intraurban transport and service, have been well described, [bib_ref] Sustainable urban forms: their typologies, models, and concepts, Jabareen [/bib_ref] and yet at the same time result in a reduction in quality of life measures. [bib_ref] The compact City fallacy, Neuman [/bib_ref] Our findings add to this debate by suggesting that retaining more wild and unstructured green space within cities is important for health. # Strengths and weaknesses This was a large international study of 233 cities within 24 countries. We were able to use high-quality, internationally comparable land cover/use data from the European Urban Atlas for each city to assess the configuration and quantity of these within each city. By including a large number of cities and countries with differing economic position, we believe our results can be generalised to similar cities globally. We were able to dissect the impact of land covers/uses both collectively (via landscape metrics) and individually. We surpass some previous research by including a very large range of land covers/uses, rather than studying the impact of just one or two.We were able to match previously validated SMR data to land cover/ use data for the same areas and a close time point. SMRs are robust, valid and widely used measures of population health. It is important to consider that land covers/uses within cities will co-occur, for example, dense compact cities may have a high proportion of both industrial and high-density residential land covers/uses. A strength of our analysis was that we assessed each land cover/use separately, allowing us to examine whether if similar types of land covers/uses were important for health. The aim of our study was not to understand the influence or configuration of combinations of land cover/use on health but nonetheless this offers an important line of enquiry for future research. Although we described associations between land covers/uses and SMRs, we have no information on how the individuals used or were exposed to the land covers/ uses that we included in our analysis, for example, if they live in a part of the city without that particular land use, and were unable to distinguish between land covers and land uses. This may be most pertinent for the most socioeconomically deprived individuals who may have a smaller activity space and therefore the affordances of the city-wide environment on their mortality may be limited. Data were aggregated to the city level and therefore may be subjected to both the ecological fallacy and modifiable areal unit problem. The development of the sample cities may also have changed considerably during individuals' life courses and the individual may not have always resided in that city, thus impacting on SMRs. As this was an ecological study and only measured land cover/use at one point in time and therefore unable to determine causality. We explored the possible effects of cultural and economic differences in land covers/uses by analysing our cities by macroregion, however we did not explore the different cultural meanings for each city to be able to ascertain if culturally affirming landscapes were health beneficial. The mortality indicator we used for the study was a city-level population-level measure and therefore we were unable to explore inequality in mortality by socioeconomic status. # Conclusion We found that the proportion of specific land covers/ uses within cities is associated with mortality rates there, but that these associations varied by macroregion and by sex. There is great pressure to build on wild green space for economic gain and sustainability, and to promote compact cities as sustainable. Our study suggests that urban planners and developers need to regard retaining more wild and unstructured green space as important for healthy city systems, and that they should reconsider the push for dense and compact cities. twitter Jonathan R Olsen @JonROlsen Contributors JRO, NN and RM contributed to the conception and design of the study. NN performed the statistical analysis. JRO performed the geospatial analysis. RM, GM, JP and NS were involved in previous work developing the main outcome measure. JRO prepared the first draft of the manuscript, with all authors contributing to its main content and revising it with critical comments. All authors have read and approved the manuscript prior to submission, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding JO, NN and RM are employed by the University of Glasgow and funded as part of the Neighbourhoods and Communities Programme (MC_UU_12017/10) (SPHSU10) at the MRC/CSO Social and Public Health Sciences Unit (SPHSU). Map disclaimer The depiction of boundaries on this map does not imply the expression of any opinion whatsoever on the part of BMJ (or any member of its group) concerning the legal status of any country, territory, jurisdiction or area or of its authorities. This map is provided without any warranty of any kind, either express or implied. Competing interests None declared. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. data availability statement Standardised Mortality Ratio data are available on reasonable request by emailing richard. mitchell@ glasgow. ac. uk. European Urban Atlas data may be obtained freely from a third party, the European Environment Agency (https://www. eea. europa. eu/ data-and-maps/ data/ urban-atlas), provided by the European Commission of the European Union. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https:// creativecommons. org/ licenses/ by/ 4. 0/. ## Orcid id Jonathan R Olsen http:// orcid. org/ 0000-0002-5356-8615 reFerenCeS [fig] Figure 1: Urban Atlas land use/cover categories and satellite base map. [/fig] [fig] Figure 2: Estimated standardised mortality ratios (SMRs) for cities with varying quintiles of agricultural, semi-natural areas, wetlands (quintile 1=lowest proportion). [/fig] [fig] Figure 3: Comparisons between standardised mortality ratio (SMR) and quintiles land covers/uses for green urban areas and industrial, commercial, public, military (partially universal importance by macroregion and/or sex). [/fig] [table] Table 1: Land covers/uses with strongest and most consistent evidence of association with city SMRs *Most evident in cities with the highest proportion of this land cover/use (quintile 5). SMR, standardised mortality ratio. [/table] [table] Table 2: Regression coefficients of land covers/uses found to contribute to a model for a particular macroregion/sex based on Wald test measure (significant results marked (<0.05) in bold font) [/table]

Comparison of cardiovascular response to sinusoidal and constant lower body negative pressure with reference to very mild whole-body heating Background:The purpose of the present study was to compare sinusoidal versus constant lower body negative pressure (LBNP) with reference to very mild whole-body heating. Sinusoidal LBNP has a periodic load component (PLC) and a constant load component (CLC) of orthostatic stress, whereas constant LBNP has only a CLC. We tested two sinusoidal patterns (30-s and 180-s periods with 25 mmHg amplitude) of LBNP and a constant LBNP with −25 mmHg in 12 adult male subjects.Results: Although the CLC of all three LBNP conditions were configured with −25 mmHg, the mean arterial pressure (MAP) results showed a significantly large decrease from baseline in the 30-s period condition (P <0.01). In contrast, the other cardiovascular indices (heart rate (HR), stroke volume (SV), cardiac output (CO), basal thoracic impedance (Z 0 ), total peripheral resistance (TPR), the natural logarithmic of the HF component (lnHF), and LF/HF (ln (LF/HF))) of heart rate variability (HRV) showed relatively small variations from baseline in the 30-s period condition (P <0.01). The result of the gain and phase of transfer function at the sinusoidal period of LBNP showed that the very mild whole-body heating augmented the orthostatic responses.Conclusion: These results revealed that the effect of the CLC of LBNP on cardiovascular adjustability was attenuated by the addition of the PLC to LBNP. Based on the results of suppressed HRV response from baseline in the 30-s period condition, we suggest that the attenuation may be caused by the suppression of the vagal responsiveness to LBNP. # Background Lower body negative pressure (LBNP) is used as a perturbation to the cardiovascular system and has been applied to simulate the gravitational stress of orthostatic blood shift in humans [bib_ref] Hinghofer-Szalkay H: LBNP: past protocols and technical considerations for experimental design, Goswami [/bib_ref] [bib_ref] Lower body negative pressure as a model to study progression to acute..., Cooke [/bib_ref]. Orthostatic faint is one of the non-adaptive responses to the gravitational stress in humans, and that has not been described in apes [bib_ref] Fainting in animals, Van Dijk [/bib_ref]. The metabolic demand of a human's large brain needs a large proportion of cardiac output (CO) to be pumped upward and the relative long leg causes the large amount of blood pooling in the leg in the orthostatic stress [bib_ref] Fainting in animals, Van Dijk [/bib_ref] [bib_ref] Brain growth, life history, and cognition in primate and human evolution, Leigh [/bib_ref] [bib_ref] Early hominin limb proportions, Richmond [/bib_ref]. It is presumed that the combination of these anatomical features of large brain and long leg in human evolution causes orthostatic difficulty. The transient phenomenon is one of the features of orthostatic faint [bib_ref] Cardiovascular regulation in the period preceding vasovagal syncope in conscious humans, Julu [/bib_ref] [bib_ref] Hinghofer-Szalkay HG: Individual stability of orthostatic tolerance response, Goswami [/bib_ref]. Oscillatory LBNP methods have been used in several studies to investigate the transient characteristics of the orthostatic response with high repeatability [bib_ref] Dynamic autoregulation of cutaneous circulation: differential control in glabrous versus nonglabrous skin, Wilson [/bib_ref] [bib_ref] Sympathetic control of the cerebral vasculature in humans, Hamner [/bib_ref] [bib_ref] Effects of heat stress on dynamic cerebral autoregulation during large fluctuations in..., Brothers [/bib_ref]. However, at present, sinusoidal LBNP methods are not major among oscillatory LBNP methods. Considering the spectral leakages of oscillatory LBNP, the waveform of LBNP should be a sinusoidal pattern during oscillatory LBNP. Although a few previous studies used sinusoidal LBNP as the perturbation stimulus [bib_ref] The repeatability of cerebral autoregulation assessment using sinusoidal lower body negative pressure, Birch [/bib_ref] [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] , only [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] showed the result of an analysis of the actual gauge pressure of sinusoidal LBNP. They investigated the frequency characteristics of hemodynamic changes induced by sinusoidal LBNP in humans across a range of 10-to 250-s periods (that is, 0.1 to 0.004 Hz) [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref]. They revealed that the cardiovascular adjustability to sinusoidal LBNP was maintained at a period slower than 50-s (that is, 0.02 Hz) oscillation. However, to our knowledge there is no previous study that directly compared sinusoidal LBNP and constant LBNP which is a conventional method in LBNP studies. Sinusoidal LBNP contains not only the periodic load component (PLC) but also the constant load component (CLC) of LBNP, whereas the constant LBNP contains only CLC (see [fig_ref] Figure 1: Traces of heart rate [/fig_ref]. CLC is the average load during LBNP, and that is a 0 Hz frequency (that is, infinite periodic) component in frequency analysis. The comparison of cardiovascular response to sinusoidal and constant LBNP under the same CLC condition could reveal the effect of PLC in sinusoidal LBNP. Orthostatic cardiovascular regulation is compromised by heat stress [bib_ref] Heat stress reduces cerebral blood velocity and markedly impairs orthostatic tolerance in..., Wilson [/bib_ref] [bib_ref] Insufficient cutaneous vasoconstriction leading up to and during syncopal symptoms in the..., Crandall [/bib_ref]. Whole-body heating during LBNP causes peripheral vasodilation, decreases cerebral blood flow (CBF), and reduces orthostatic tolerance. Warm environments are described as one of the most common triggers for fainting episodes in the general population [bib_ref] Lifetime cumulative incidence of syncope in the general population: a study of..., Ganzeboom [/bib_ref]. Although there is considerable inter-individual variation in orthostatic tolerance among normal subjects [bib_ref] Hinghofer-Szalkay HG: Individual stability of orthostatic tolerance response, Goswami [/bib_ref] [bib_ref] Error and individual difference in cardiovascular responses to orthostatic stress in humans, Ishibashi [/bib_ref] , evidence that fainting has a genetic basis is not very strong [bib_ref] Heterogeneity of responses to orthostatic stress in homozygous twins, O&apos;leary [/bib_ref] [bib_ref] Genetic aspects of vasovagal syncope: a systematic review of current evidence, Nordkamp [/bib_ref] [bib_ref] Lack of association between genetic polymorphisms affecting sympathetic activity and tilt-induced vasovagal..., Sorrentino [/bib_ref]. To elucidate the explanatory variables accounting for the large inter-individual variation in physiological responses, the relationship between reduced orthostatic tolerance in warm environments and the broad range of lifestyle habits such as habitual physical exercise and chronobiological rhythms of the subjects could also be investigated. Sedentary and evening type lifestyles differ with regard to their cardiovascular responses to stress [bib_ref] Of larks and hearts-morningness/eveningness, heart rate variability and cardiovascular stress response at..., Roeser [/bib_ref] [bib_ref] Effects of 12 weeks of physical training on cardiovascular responses to head-up..., Aoki [/bib_ref]. In hindsight, although we used very mild whole-body heating in this study, the very mild hyperthermia effect on orthostatic cardiovascular response to sinusoidal and constant LBNP was investigated. Accordingly, the first objective of the present study was to investigate the effects of CLC and PLC of LBNP with a comparison of the cardiovascular responses to sinusoidal LBNP and constant LBNP with reference to very mild whole-body heating. In light of the finding by [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] that a 50-s period was the boundary period of the AP regulation to sinusoidal LBNP, we tested two different periods (30 s and 180 s) of the PLC of LBNP. Our second objective was to investigate the relationship between the response to the LBNP and lifestyle habits of the subject, as an attempt to identify any explanatory variables for the large interindividual variation in orthostatic tolerance. )) participated in the study. No subjects smoked or took medication regularly, and they refrained from alcohol consumption and heavy exercise for at least 24 h prior to participating in the study. The study was approved by the Research Ethics Committee of the Chiba University Faculty of Engineering . Written informed consent was obtained from all subjects before the start of the experiment. # Methods ## Subjects ## Lbnp We used the electronically controlled LBNP system that we devised and reported previously; the technical issues regarding the construction of the LBNP chamber and the electronic control of the LBNP are as described [bib_ref] Construction of an electronic controlled lower body negative pressure chamber using commercial..., Ishibashi [/bib_ref]. To improve the accuracy of the LBNP control, we installed a differential pressure gauge (PU-10kPa; Halstrup-Walcher, Kirchzarten, Germany) and an electronically controlled blower (VASF 1.50/1; Gebr Becker, Wuppertal, Germany) in the LBNP system. The controllable range of the pressure gauge of this LBNP system was from −0.60 to −60.00 mmHg with a 10-bit DA controller. Therefore, the air pressure from 0 (that is, sea level) to −0.60 mmHg was out of range of the controller. Two sinusoidal patterns of LBNP and a constant LBNP were applied. The periods of sinusoidal curve were 30 s (0.033 Hz) and 180 s (0.0055 Hz). The range of the curve was configured using 0 to −50 mmHg. The constant LBNP level was set at −25 mmHg [fig_ref] Figure 1: Traces of heart rate [/fig_ref]. The duration of the LBNP was 12 min. The baseline period was 6 min before LBNP. The average gauge pressures that were the PLC of all three LBNP conditions were set at −25 mmHg. ## Experimental protocol The experimental protocol is shown in [fig_ref] Figure 2: Experimental protocol [/fig_ref]. The experiments were carried out in a semi-dark thermoneutral room (temperature, 27.25°C ± 0.29°C (SE) (range, 26.62°C to 27.88°C); relative humidity, 52.2 ± 0.4% (SE) (range, 51.2% to 53.1%)). The subjects reported to the experimental room at 10:00 (n = 6), 12:00 (n = 1), or 16:00 (n = 5). Each subject's preparation for the experiment started approximately 1 h prior to the start of the measurement: the subject emptied his bladder, had the electrode and thermistors attached. The subject exchanged his clothing for a long-sleeved T-shirt (KZ3002, Gunze, Kyoto, Japan) and long-johns (KZ3002, Gunze) to prevent low-temperature injury from the direct heating by the tube-line suit. Under the long-sleeved T-shirt, a neoprene skirt (KAKR002, Sandiline, Koper, Slovenia) for airtight sealing was fitted around the subject's iliac crest. Over his clothes and neoprene skirt, the subject wore a waterperfused tube-lined suit (shirt, 420-122; pants, 420-126, Med-Eng, Allen-Vanguard Corp., Ottawa, Canada) and was placed inside the LBNP chamber in the supine position. A saddle in the LBNP chamber provided perineal support and prevented caudal displacement of the subject. The subject was allowed to watch mildly stimulating movies were projected on the chamber's ceiling to counteract any sleepiness. The water temperature was adjusted by a temperaturecontrolled bath (BQ100, Yamato Scientific Co., Tokyo, Japan). For the separate water circulation of the upper and lower parts of the tube-lined suit, two pumps were used (Delta Wing Pump, Allen-Vanguard Corp.). Three different LBNP conditions were measured in random order in each thermal condition. The inter-test interval was set at 23 min: a 6-min baseline, 12-min LBNP, and 5-min break time (see [fig_ref] Figure 2: Experimental protocol [/fig_ref]. The water temperature for the water-perfused tube-lined suit for thermoneutral condition was set at 32°C. For the whole-body heating condition, the water temperature was increased to 42°C within 30 min after the thermoneutral condition was confirmed. After the 30-min heating period, three different LBNP conditions during whole-body heating were measured. ## Data acquisition and analysis Temperature data were recorded by a data logger (LT-8A; Gram Corp., Saitama, Japan) at 2-s intervals. Rectal temperature (Tre) was measured by a thermistor (LT-ST08-11; Gram) with a disposable rubber sheath (RC5020-A; Nikkiso-Therm Co. Tokyo) inserted 12 cm into the rectum. Skin temperatures (Tsk) were also measured using a thermistor (LT-ST08-00; Gram) fixed to the skin with adhesive surgical tape (Surgical Tape-21 N; Nichiban Co. Tokyo, Japan). Skin temperatures were measured at the forehead (Tsk-head), abdomen (Tskabdomen), and dorsum of the left foot (Tsk-foot). These three skin-temperature measuring points were not covered by the water-perfused tube-lined suit. The weighting coefficient for the mean skin temperature from these three points was not clear, and therefore the mean skin temperature was not calculated. The water temperature of the temperature-controlled bath and the air temperature in the LBNP chamber were also measured using a thermistor (LT-ST08-00; Gram). The water temperature was 31.96°C ± 0.01°C (SE) (range, 31.93°C to 32.01°C) during the thermoneutral condition, and 41.88°C ± 0.06°C (SE) (range, 41.25°C to 42.03°C) during the whole-body heating condition. ECG, impedance-cardiogram (ICG), and phonocardiogram (PCG) signals were taken using a polygraph system (AB-621 G for ECG, AI-601 G and ED-601 G for ICG, and AS-601H for PCG; Nihon Kohden Corp., Tokyo, Japan) and were recorded using a personal computer with an analog-to-digital conversion rate of 1 kHz per channel by a 16-bit AD converter (AD16-16U(PCI)EH; Contec Co., Osaka, Japan). From the ICG signal, the basal thoracic impedance (Z 0 ), the delta impedance waveform (ΔZ), and its first derivative (dZ/dt) were derived. Z 0 is inversely related to central blood volume [bib_ref] The use of thoracic impedance for determining thoracic blood volume changes in..., Ebert [/bib_ref]. SV values were estimated by Kubicek's method using the dZ/dt signal [bib_ref] Development and evaluation of an impedance cardiac output system, Kubicek [/bib_ref]. Blood resistivity (ρ) was set at 135 cm · ohm. To estimate the SV on a beat-by-beat basis, the noise of the dZ/dt signals was eliminated by an adaptive filter that can select the components that are synchronous with the R-R interval (RRI) of the ECG [bib_ref] Filtering noncorrelated noise in impedance cardiography, Barros [/bib_ref]. The same filter was applied to derive the respiration curve from ΔZ [bib_ref] Modified thoracic impedance plethysmography to monitor sleep apnea syndromes, Yasuda [/bib_ref]. The beat-by-beat heart rate (HR) was calculated from the RRI sequences of the ECG. CO was also calculated (= SV × HR) on a beat-by-beat basis. AP was measured intermittently by a non-invasive oscillometric blood pressure monitor (HEM-7200; Omron Healthcare, Kyoto, Japan). AP was not measured continuously because of the limitations of our experimental facility. The measuring interval was set at approximately 100 s to avoid the biased distribution of the measuring timing in the sinusoidal phase of LBNP. Therefore, at each measuring interval, the lag angle against the sinusoidal LBNP for the start timing of cuff inflation was shifted 120°a nd 200°for the 30-s and 180-s conditions, respectively. The AP value was averaged for the baseline and for the LBNP period. The TPR was calculated by dividing the mean arterial pressure (MAP) by the CO. Therefore, the periodic responses of MAP and TPR cannot be described in this study. The first 3 min of data in the LBNP period were not used for the analysis, to ensure the stabilization of the physiological condition of the subject. The last 3 min of data of in the LBNP period were also eliminated from the analysis, to align the same data length to 6-min baseline period data. The unequal intervals of the beat-bybeat data of RRI, SV, and CO were interpolated into 5.689-Hz (= 1,024 point/180 s) equidistant data. The 6min RRI dataset, with the Hanning window after linear trends were eliminated by linear regression, was used for the HRV analysis. The coarse graining spectral analysis (CGSA), the algorithm developed by Yamamoto and Hughson [bib_ref] Extracting fractal components from timeseries, Yamamoto [/bib_ref] , was applied for the HRV analysis. The advantages of using the CGSA for an HRV analysis were described [bib_ref] Inhibition of heart rate variability during sleep in humans by 6700 K..., Ishibashi [/bib_ref]. The fractal-free high-frequency (HF) and low-frequency (LF) components were integrated from 0.15 to 0.50 Hz and from 0.05 to 0.15 Hz, respectively, of the power spectra. The HF components of HRV are considered markers of cardiac vagal activity, whereas the LF components are markers of both cardiac vagal and sympathetic activities [bib_ref] Assessment of autonomic function in humans by heart rate spectral analysis, Pomeranz [/bib_ref]. The LF/HF ratio was calculated for the index of sympathovagal balance [bib_ref] Power spectral analysis of heart rate and arterial pressure variabilities as a..., Pagani [/bib_ref]. To normalize the distribution of HRV parameters, we used the natural logarithmic transformed values for the analysis (that is, the natural logarithmic of the LF component of HRV (lnLF), the natural logarithmic of the HF component of HRV (lnHF), and the natural logarithmic ratio of LF/HF of HRV, or 'ln(LF/HF)'). The advantages of the natural logarithmic transformation in HRV were thoroughly explained in analyses of the data of relatively large numbers of subjects [bib_ref] Normative references of heart rate variability and salivary alpha-amylase in a healthy..., Kobayashi [/bib_ref]. To check the contamination of the respiratory component in the LF component of HRV, we also calculated the percent LF to total power (TP; 0.00-0.50 Hz) of the respiration curve from ΔZ. The transfer function data between the cardiovascular indices (RRI, SV, CO, and Z 0 ) and the sinusoidal gauge pressure of LBNP were estimated from the same time period of the dataset of the HRV analysis. Gain, phase, and coherence during the LBNP period were derived from the cross-spectral analysis on a fast Fourier transformation basis. These transfer function data were analyzed only during sinusoidal LBNP. The baseline period and the constant LBNP condition were excluded from the transfer function analysis. A questionnaire regarding lifestyle habits was completed by each subject. The questionnaire gathered information about the subject's habitual physical exercise per week, average exposure time to air-conditioned rooms per day, and the frequency of going without breakfast [bib_ref] Effects of lifestyle, body composition, and physical fitness on cold tolerance in..., Maeda [/bib_ref]. The Japanese version of the Morningness-Eveningness Questionnaire (MEQ) score by Horne and Östberg [bib_ref] A self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms, Horne [/bib_ref] regarding chronobiological rhythms was also completed by each subject. # Statistical analysis The effects of heat stress ('Heating' , or thermoneutral and whole-body heating) on the mean values during the baseline were analyzed with a repeated-measures analysis of variance (ANOVA). To check the order effect in each thermal condition as a confounding factor on baseline values, three conditions of LBNP order ('Order': LBNP1, LBNP2, and LBNP3) and three conditions of LBNP ('Period': 30-s period, 180-s period, and constant) were also included in the factors of ANOVA. The difference of mean values from baseline and LBNP were analyzed with a two-way ANOVA followed by a simple effect test with Bonferroni correction for the post-hoc analysis (SPSS Inc., Chicago, IL, USA). The factors were two levels of heat stress (Heating) and three levels of LBNP condition (Period). For the transfer function data of gain, phase, and coherence, the effects of heat stress ('Heating': thermoneutral and whole-body heating) and two conditions of LBNP ('Period': 30-s period and 180-s period) were analyzed by a two-way repeated-measures ANOVA followed by a simple effect test with Bonferroni correction for the post-hoc analysis (SPSS). The units of the phase (degree) and coherence (coefficient) were identical in the cardiovascular variables. The comparison of variables was performed with a repeated-measures ANOVA. The Greenhouse-Geisser correction was used to evaluate P values for repeated measures involving more than one degree of freedom. Greenhouse-Geisser P values were based on corrected degrees of freedom, but the original degrees of freedom are reported. A linear regression analysis was used to examine the relationship between the cardiovascular index and the results of the lifestyle habits questionnaire. To analyze differences in the regression coefficients (that is, the slope of the regression line) among the heating conditions, we used an analysis of covariance (ANCOVA) [bib_ref] The Iowa State University Press: 6th edn, Snedecor [/bib_ref]. All data are expressed as mean ± SE, and the level of significance was set at P < 0.05. # Results ## Physical data The average gauge pressure (CLC) of LBNP was set at −25 mmHg in all conditions. There were marginal but significant differences in the gauge pressure among the LBNP conditions The air temperature in the LBNP chamber was significantly affected by heating condition (thermoneutral, 30.00 ± 0.10°C; whole-body heating, 32.15 ± 0.07°C; F(1,11) = 755.22, P <0.001) and by LBNP (baseline, 31.53 ± 0.10°C; LBNP, 30.62 ± 0.07°C; F(1,11) = 209.94, P <0.001). The heating-condition effect was caused mainly by the convection and the radiation of heat from the water-perfused tube-lined suit, and the LBNP effect was caused mainly by Charles's law. ## Baseline data The results of baseline values under the thermoneutral and heating conditions are shown in [fig_ref] Table 1: Mean values of baseline under the thermoneutral and heating condition, and the... [/fig_ref]. Wholebody heating significantly increased the baseline values of Tre (P <0.05), Tsk-abdomen (P <0.001), Tsk-foot (P <0.05), HR (P <0.05), TPR (P <0.05), and ln(LF/HF) of HRV (P <0.01), but decreased the SV (P <0.01), CO (P <0.05) and lnHF of HRV (P <0.01). As for the order effect (Order), there was a significant interaction between Order and Heating condition at baseline in Tre (thermoneutral, -0.12 ± 0.02°C (LBNP1 vs. LBNP3) and whole-body heating, +0.14 ± 0.03°C (LBNP1 vs. LBNP3); F(2,22) = 47.37, P <0.001. However, the main effect of LBNP condition (Period) and the interaction between Period and Heating condition were not significant in the baseline values of all variables. Therefore, the order effect as the confounding factor was randomized and minimized among the LBNP conditions. ## Response to lbnp of hemodynamic data The differences in mean values from baseline to LBNP in each condition are shown in [fig_ref] Figure 3: Differences in mean values [/fig_ref]. The main effect of the Heating was significant only for HR (P <0.001). Whole-body heating augmented the increase in HR during LBNP. There were significant main effects of Period in HR (P <0.001), SV (P <0.001), CO (P <0.001), Z 0 (P <0.001), MAP (P <0.01), and TPR (P <0.001). The simple main effect of the Period condition showed that the effect of LBNP was unique in MAP. The other variables showed a relatively small variation in the 30-s period condition compared to the 180-s period and constant LBNP conditions, whereas the MAP results showed a large drop in the 30-s period condition. ## Response of the hrv data to lbnp The contamination of the respiratory component in the LF band was negligible. The ratio of LF band power to TP of the respiration curve was 1.46 ± 0.39%. The spectral leakages of gauge pressure of sinusoidal LBNP in the LF and HF bands were also negligible. The ratios of LF and HF to TP were 0.022 ± 0.005% (LF band) and 0.010 ± 0.002% (HF band), respectively. [fig_ref] Figure 3: Differences in mean values [/fig_ref] shows the differences in mean values from baseline to LBNP of the lnLF, lnHF, and ln(LF/HF). There were significant main effects of Period in lnHF (P <0.01) and ln(LF/HF) (P <0.01). The simple main effect of the Period condition showed that the effect of LBNP was relatively large in the constant LBNP condition in both lnHF and ln(LF/HF). Transfer function data [fig_ref] Figure 4: Results of transfer function data between LBNP gauge pressure [/fig_ref] shows the results of transfer function data between LBNP gauge pressure and HR, SV, CO, and Z 0 of gain [fig_ref] Figure 4: Results of transfer function data between LBNP gauge pressure [/fig_ref] , row A), phase (row B), and coherence (row C) in the LBNP period and the result of the repeated-measures two-way ANOVA. Regarding the transfer gain results, the main effect of the Heating was significant in HR (P <0.001), SV (P <0.05), and Z 0 (P <0.05). The gain reflects the amplitude of the oscillation at the LBNP period. In the whole- body heating condition, large amplitudes of HR, SV, and Z 0 were observed. The main effect of the Period was significant in HR (P <0.01), SV (P <0.001), CO (P <0.001), and Z 0 (P <0.001) with relative small variation in the 30-s period condition compared to the 180-s period. There was a significant interaction between the Heating and Period in CO (P <0.05). The simple main effect showed that the effect of the Heating was significant in the 30-s period condition (P <0.05) but not in the 180-s period condition. Regarding the transfer phase results, the values of HR and Z 0 indicated relative large phase angles compared to SV and CO. These large difference between the parameters were reflecting the opposite response direction to LBNP. The HR and Z 0 increased during LBNP, but the SV and CO decreased [fig_ref] Figure 1: Traces of heart rate [/fig_ref]. There was a significant main effect of the Heating in HR. The positive phase angle reflects the delay of the oscillation to the sinusoidal LBNP. Whole-body heating decreased the lag angle of HR. However, the main effect of Period was significant in all variables (HR, SV, CO, and Z 0 , P <0.001 for all variables). The lag angle to the sinusoidal LBNP was relatively large in the 30-s period condition. The comparison of variables revealed that phase was significantly different between the variables (F(3,33) = 686.04, P <0.001). The simple main effect showed that the lag angle in HR (P <0.01) was relatively large compared to that in Z 0 . Regarding the transfer coherence results, the main effect of Heating was significant in SV (P <0.05). The coherence reflects the similarity of the oscillation to the sinusoidal LBNP. Under the whole-body heating condition, relatively large coherence was shown in SV. The main effect of Period was significant in Z 0 (P <0.001) with relatively small coherence in the 180-s period condition compared to the 30-s period. # Linear regression analysis The linear regression analysis examining the cardiovascular index and the results of the lifestyle habits questionnaire showed that the correlation of HR and MEQ score was significant. The habitual physical exercise times per week, the average exposure time to an airconditioned room per day, and the frequency of going without breakfast were not significantly correlated with the cardiovascular index. Moreover, the significant correlation between HR and MEQ score was shown in the 180-s period and the constant LBNP condition (P <0.05 for both conditions), but not in the 30-s period LBNP condition [fig_ref] Figure 5: Linear regression analysis of the inter-individual relationship between the morningness-eveningness questionnaire [/fig_ref]. The subjects with higher Morningness scores tended to adjust to the 180-s period and constant LBNP by a small increase in HR. The correlation coefficient between start time of measurement and MEQ score was not significant (r = −0.174, P = 0.588, n.s.). When the ANCOVA was performed on HR and MEQ score, there were no significant differences in regression coefficients between the Heating conditions in the 180-s period (F(1,20) = 0.60, n.s.) and the constant LBNP condition (F(1,20) = 0.46, n.s.). # Discussion The major finding of this study was that the cardiovascular responses, except the MAP, were attenuated in the sinusoidal (PLC plus CLC) LBNP compared to the constant (CLC only) LBNP, although the average gauge pressure (CLC) during the LBNP period was set at −25 mmHg in all conditions. The mean Z 0 values showed that the degree of the thoracic blood volume shift to LBNP was the largest in the constant LBNP condition, second largest in the 180-s period condition, and the smallest in the 30-s period condition [fig_ref] Figure 3: Differences in mean values [/fig_ref]. Z 0 is inversely related to central blood volume [bib_ref] The use of thoracic impedance for determining thoracic blood volume changes in..., Ebert [/bib_ref]. The order of the degree of the variation against each LBNP condition was the same in the decreases of the SV and CO [fig_ref] Figure 3: Differences in mean values [/fig_ref] and in the rise of the TPR [fig_ref] Figure 3: Differences in mean values [/fig_ref]. As for the orthostatic regulation of blood pressure, the MAP results showed a large drop in the 30-s period condition [fig_ref] Figure 3: Differences in mean values [/fig_ref] , whereas the other cardiovascular indices showed relative small variation in that condition. These results confirmed the previous report [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] that the cardiovascular adjustability to sinusoidal LBNP was maintained at the period of slower than 50-s (that is, 0.02 Hz) oscillation. [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] revealed that cardiovascular adjustability to sinusoidal LBNP was maintained >50 s of the period of oscillation with an amplitude of 25 mmHg of LBNP. The amplitude of 25 mmHg of sinusoidal LBNP has a CLC of −25 mmHg. However, to our knowledge there is no previous study that compares sinusoidal LBNP with constant LBNP. The results of the present study revealed that the effect of the CLC of LBNP on cardiovascular adjustability was attenuated by the addition of PLC to LBNP. Considering that the spectral leakages from the main periodic component of oscillatory LBNP directly contaminate the other spectral components, the waveform of LBNP should be a sinusoidal pattern in cases of simultaneous measurements of HRV during oscillatory LBNP. In the present study's HRV results, the response of lnHF and ln(LF/HF) to LBNP were attenuated in the sinusoidal LBNP compared to the constant LBNP. Since there was no significant effect on lnLF, the ln(LF/HF) results reflect a dominant effect of lnHF, which is an index of vagal activity [bib_ref] Assessment of autonomic function in humans by heart rate spectral analysis, Pomeranz [/bib_ref]. Therefore, we surmise that the attenuated cardiovascular adjustability by the addition of periodic oscillation of LBNP was caused by the suppression of the vagal responsiveness to LBNP. Regarding the whole-body heating, the baseline of Tre was raised by almost 0.1°C by this experimental protocol. Previous studies showed the increase of their subjects' core temperature from 0.5°C to 1.5°C by a water perfusion suit with a relatively higher water temperature [bib_ref] Effects of heat stress on dynamic cerebral autoregulation during large fluctuations in..., Brothers [/bib_ref] [bib_ref] Insufficient cutaneous vasoconstriction leading up to and during syncopal symptoms in the..., Crandall [/bib_ref]. Although the heating condition in the present study was very mild whole-body heating, the significant main effect of the Heating on baseline values was observed for Tre, Tsk-abdomen, Tsk-foot, HR, SV, CO, TPR, lnHF, and ln(LF/HF). We suspect that the significant main effect of the Heating on ln(LF/HF) is the result of relative sympathetic activation during wholebody heating. However, there was no significant effect on lnLF. Moreover, there is an argument that the LF of HRV is not a biomarker of sympathetic activity but rather is a measure of the modulation of cardiac autonomic outflows by the baroreflex [bib_ref] Low-frequency power of heart rate variability is not a measure of cardiac..., Goldstein [/bib_ref]. Generally, heat stress does not alter the baroreflex control of the heart [bib_ref] Cardiovascular function in the heatstressed human, Crandall [/bib_ref] , and therefore, the present study's finding on lnLF could be adequate. The baseline values of lnHF and SV dropped significantly during the Heating condition. The decreases in HF of HRV and SV are known to indicate a reduction in venous return [bib_ref] Hypothesis: respiratory sinus arrhythmia is an intrinsic resting function of cardiopulmonary system, Hayano [/bib_ref]. However, since the CO baseline values were decreased by the heating although MAP was maintained in our study, TPR was estimated that was increased in the heating condition. In previous studies using whole-body heating, the TPR dropped markedly with heating [bib_ref] Cardiovascular responses to sustained high skin temperature in resting man, Rowell [/bib_ref]. However, heat stress induces vasoconstriction in non-cutaneous beds (that is, splanchnic, renal, muscle, and cerebral) [bib_ref] Heat stress reduces cerebral blood velocity and markedly impairs orthostatic tolerance in..., Wilson [/bib_ref] [bib_ref] Human cardiovascular adjustments to exercise and thermal stress, Rowell [/bib_ref]. The present finding of a relatively small increase in TPR might reflect non-cutaneous vasoconstrictions caused by the very mild whole-body heating. We should have measured cutaneous vascular resistance (CVR) in reference to the TPR. As for the mean values' responses to LBNP, the ANOVA showed that the main effect of Heating was significant only in HR [fig_ref] Figure 3: Differences in mean values [/fig_ref]. However, in the gain of the transfer function, the significant main effect of the Heating was observed in HR, SV, and Z 0 [fig_ref] Figure 4: Results of transfer function data between LBNP gauge pressure [/fig_ref]. As for the influence of temperature on the distribution of blood, which was examined by right heart catheterization [bib_ref] Effect of thermal stress on Frank-Starling relations in humans, Wilson [/bib_ref] , the measurement of Z 0 [bib_ref] Influence of temperature on the distribution of blood in humans as assessed..., Cai [/bib_ref] , and gamma camera imaging [bib_ref] Colloid volume loading does not mitigate decreases in central blood volume during..., Crandall [/bib_ref] , heat stress induces a central blood volume reduction. Although the heating conditions used in the present study were very mild compared to those of previous studies, we were able to detect the significant increase in the gain of Z 0 in the Heating condition. Considering that the beat-by-beat signal of the cardiovascular response contains other periodic fluctuations (that is, respiratory sinus arrhythmia and Mayer wave-related sinus arrhythmia), a transfer function analysis of a target sinusoidal period of LBNP would be advantageous to experimentally detect cardiovascular adjustability, with high repeatability. Regarding the phase results and the transfer function, a significant main effect of the Period was observed in all variables (HR, SV, Co, and Z 0 ; see [fig_ref] Figure 4: Results of transfer function data between LBNP gauge pressure [/fig_ref]. These results confirmed those of [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref] , who showed the relatively large lag angle to the sinusoidal LBNP in a short period of condition. The phase angle of HR and Z 0 were relatively large compared to SV and CO, reflecting the adverse response direction to LBNP. Within the variables of the same response direction to LBNP, the lag angle in HR was relatively large compared to Z 0 , reflecting the cascade reaction to LBNP (that is, the reduction of central blood volume could be preceding the HR response to LBNP). Moreover, the significant main effect of the Heating on the HR of phase of the transfer function indicated that the lag angle in the HR was decreased by heat stress. Considering the augmented gain in HR caused by whole-body heating, the HR response to LBNP in heat stress was characterized by a large amplitude and quick reaction. In this study, the AP was not measured continuously because of the limitations of our experimental facility. The dynamic responses of AP, TPR, and also CVR to sinusoidal LBNP should be investigated to increase our understanding of orthostatic intolerance under heat stress. Our finding of distinctly high coherence of Z 0 reflected the similarity to the sinusoidal LBNP. Z 0 is inversely related to central blood volume [bib_ref] The use of thoracic impedance for determining thoracic blood volume changes in..., Ebert [/bib_ref]. The similarity was close to a coefficient of 1.0 [fig_ref] Figure 4: Results of transfer function data between LBNP gauge pressure [/fig_ref]. The main effect of Period on the Z 0 of phase indicated that the coherence in Z 0 was relatively lower in the 180-s period condition compared to the 30-s period. This might not have been caused as a physiological consequence, because the set value of the 180-s period of sinusoidal LBNP curve has a relatively long duration to maintain above −0.6 mmHg, which was the upper limit of the controllable range of gauge pressure of the LBNP system we used. Nevertheless, sinusoidal LBNP caused a sinusoidal thoracic blood shift. As for the association between the subjects' lifestyle habits and their cardiovascular responses to LBNP, the linear regression analysis showed a significant correlation between HR and MEQ score, except for the 30-s period LBNP condition. The subjects' dietary habits and daily physical activity were not significantly correlated with their cardiovascular responses to LBNP. Previous studies showed that individual differences in MEQ score were related to the subjects' baseline HR [bib_ref] Of larks and hearts-morningness/eveningness, heart rate variability and cardiovascular stress response at..., Roeser [/bib_ref] , sleeping behavior [bib_ref] Relation of chronotype to sleep complaints in the general Finnish population, Merikanto [/bib_ref] , personality [bib_ref] Associations between chronotypes, psychopathology, and personality among incoming college students, Hsu [/bib_ref] , and mental health [bib_ref] Chronotype differences in suicidal behavior and impulsivity among suicide attempters, Selvi [/bib_ref]. The previously reported diurnal variation in vascular function indicates that the vasoconstrictor response is lower in the morning than afternoon [bib_ref] Is there diurnal variation in initial and delayed orthostatic hypotension during standing..., Lewis [/bib_ref] [bib_ref] Diurnal variation in vascular function: role of sleep, Jones [/bib_ref]. The reduced vascular function could cause the higher cardiac responsiveness to maintain the AP against the LBNP. Although high HR responsiveness is not directly correlated with LBNP tolerance [bib_ref] Hinghofer-Szalkay H: LBNP: past protocols and technical considerations for experimental design, Goswami [/bib_ref] , we suspect, but have not proved, that the reduced physiological arousal in evening-type subjects might contribute to the high HR responsiveness to the LBNP. A previous study reported that higher HR values with a low MEQ score (Evening type) was associated with low vagal activity [bib_ref] Of larks and hearts-morningness/eveningness, heart rate variability and cardiovascular stress response at..., Roeser [/bib_ref]. The present study has several limitations. There were marginal but significant differences in the gauge pressure between the LBNP conditions. The differences of 0.40 mmHg (30-s vs. constant) and 0.27 mmHg (30-s vs. 180-s) were both significant. The magnitude of LBNP is directly related to the reductions of CVP [bib_ref] Lower body negative pressure as a model to study progression to acute..., Cooke [/bib_ref] [bib_ref] Cardiovascular regulation in humans in response to oscillatory lower body negative pressure, Levenhagen [/bib_ref]. These data suggest 2-mmHg decreases of CVP for every −10 mmHg LBNP. The physiological significance of the difference of 0.40 mmHg of LBNP is not known, but it might be negligible. These marginal but significant differences may have been caused by the combination of accurate repeatability of the sinusoidal LBNP and the mismatch of PID parameters in the LBNP control system. This issue merits further study. # Conclusions In conclusion, the effect of the CLC of LBNP on cardiovascular adjustability was attenuated by the addition of the PLC of LBNP. In light of the results of the simultaneous measurements of HRV, we suggest that the attenuation may be caused by the suppression of the vagal responsiveness to LBNP. We observed that the short period of PLC decreased the response in HR, SV, CO, and TPR, but increased the response in MAP under the same CLC condition. Moreover, these PLC effects were augmented by very mild whole-body heating. Further studies to investigate the association between the broad range of lifestyle habits and the large inter-individual variation in orthostatic tolerance under heat stress are warranted. [fig] Figure 1: Traces of heart rate (HR), basal thoracic impedance (Z 0 ), and gauge pressure of LBNP from three conditions of LBNP (−25 mmHg constant LBNP, 30-s and 180-s period of sinusoidal LBNP) during thermoneutral and whole-body heating conditions in the same male subject. The average value of LBNP (CLC) was configured with −25 mmHg in all conditions. [/fig] [fig] Figure 2: Experimental protocol. [/fig] [fig] F: (1,11) = 0.02 (2, 22) = 14.30 ** (2,22) [/fig] [table] Table 1: Mean values of baseline under the thermoneutral and heating condition, and the result of ANOVA [/table]

Design of Artificial Enzymes Bearing Several Active Centers: New Trends, Opportunities and Problems # Introduction Humankind demands that chemical production should be as sustainable and clean as possible [bib_ref] Green Chemistry: Principles and Practice, Anastas [/bib_ref] [bib_ref] Fundamentals of green chemistry: Efficiency in reaction design, Sheldon [/bib_ref] [bib_ref] Green chemistry oriented organic synthesis in water, Simon [/bib_ref] [bib_ref] E factors, green chemistry and catalysis: An odyssey, Sheldon [/bib_ref] , and catalysis is a necessary tool to reach this goal. Biocatalysis should have a prominent role in this target [bib_ref] Biocatalysis using immobilized enzymes in continuous flow for the synthesis of fine..., Thompson [/bib_ref] [bib_ref] Biocatalysis and green chemistry, Sheldon [/bib_ref] [bib_ref] Biocatalysis for green chemistry and drug development, Zhenming [/bib_ref]. Enzymes are able to catalyze the most complex processes under very mild conditions in a very atom-efficient way [bib_ref] Industrial biocatalysis today and tomorrow, Schmid [/bib_ref] [bib_ref] Dispelling the myths-Biocatalysis in industrial synthesis, Schoemaker [/bib_ref]. This is due to their high activity under atmospheric pressure and room temperature, high specificity (that permits the modification of a single compound in a mixture of very similar compounds) and selectivity (that permits the production of a single product among several possible ones). Nature provides a huge amount of enzymes bearing different catalytic features, and the current development of metagenomics tools has enlarged the range of available enzymes reaching even those produced from non-cultivable or no longer existing organisms . Enzymes may be further improved to enhance their performance by genetic tools, such as directed evolution or site-directed mutagenesis (based in dynamic simulations, enzyme modelling, etc.) . Finally, enzyme chemical modification or immobilization [33-38] may be the last opportunity of making enzyme features fit specific industrial requirements. The design of artificial metalloenzymes is a quite mature discipline nowadays [bib_ref] Engineered and artificial metalloenzymes for selective C-H functionalization, Ren [/bib_ref] [bib_ref] Artificial metalloenzymes: The powerful alliance between protein scaffolds and organometallic catalysts, Large [/bib_ref] [bib_ref] Remediation of environmentally hazardous organophosphates by artificial metalloenzymes, Serafim [/bib_ref]. These artificial enzymes have special relevance when there are no natural enzymes available to catalyze the target reaction. It enables the preparation of protein-based biocatalysts where the metallic component is the catalytically active phase, whereas the enzyme environment may tune its features (providing higher catalytic efficiency, specificity or selectivity) [bib_ref] The protein environment drives selectivity for sulfide oxidation by an artificial metalloenzyme, Rousselot-Pailley [/bib_ref] [bib_ref] Unravelling novel synergies between organometallic and biological partners: A quantum mechanics/molecular mechanics..., Ortega-Carrasco [/bib_ref] [bib_ref] Artificial metalloenzymes: Combining the best features of homogeneous and enzymatic catalysis, Pordea [/bib_ref] [bib_ref] Supramolecular interactions between functional metal complexes and proteins, Davies [/bib_ref]. One step further in the complexity of biocatalytic processes is the design of multienzyme processes, which may include cascade reactions [bib_ref] Multi-enzymatic cascade reactions: Overview and perspectives, Ricca [/bib_ref] [bib_ref] Cell-free metabolic engineering: Production of chemicals by minimized reaction cascades, Guterl [/bib_ref] [bib_ref] Multienzyme cascade reactions-Status and recent advances, Sperl [/bib_ref] [bib_ref] Multi-enzymatic synthesis, Lopez-Gallego [/bib_ref] [bib_ref] Wiring step-wise reactions with immobilized multi-enzyme systems, Velasco-Lozano [/bib_ref] or the modification of several components of a complex substrate (e.g., hydrolysis of galactose and casein in milk or multiple glycerides contained in oils [bib_ref] One pot use of combilipases for full modification of oils and fats:..., Arana-Peña [/bib_ref]. These cascade processes include the step by step or simultaneous use of several enzymes. The simultaneous use of several enzymes is preferred, as this reproduces the biological metabolic chains where the enzymes work in vivo, and may be used to shift equilibrium and to reduce inhibitions or inactivations [bib_ref] Multi-enzymatic cascade reactions: Overview and perspectives, Ricca [/bib_ref] [bib_ref] Cell-free metabolic engineering: Production of chemicals by minimized reaction cascades, Guterl [/bib_ref] [bib_ref] Multienzyme cascade reactions-Status and recent advances, Sperl [/bib_ref] [bib_ref] Multi-enzymatic synthesis, Lopez-Gallego [/bib_ref] [bib_ref] Wiring step-wise reactions with immobilized multi-enzyme systems, Velasco-Lozano [/bib_ref]. Moreover, this permits the development of one step/one reactor processes, with a corresponding savings in time and resources. However, this may raise some additional problems: the reaction conditions must fit the operational window for all involved enzymes; if some deleterious product is released by one of the enzymes (e.g., hydrogen peroxide), all enzymes will be exposed to it, etc. [bib_ref] Enzyme co-immobilization: Always the biocatalyst designers' choice . . . or not?, Arana-Peña [/bib_ref]. Thanks to the huge developments in the diversity of areas involved in the design of enzymes, one of the current trends in performing cascade reactions is the design of artificial enzymes bearing two active centers. This strategy has a distinct advantage. A single peptide chain, produced in a single fermentation, may present the activities required to catalyze the whole reaction chain. This way, just one biocatalyst needs to be produced, purified and immobilized, with the corresponding cost efficiency. The conceptual advances and advantages of these enzymes bearing multiple active centers are obvious, and the potential of some of these strategies (some of them really novel) still need to be properly developed to be fully exploited. However, this can also pose some difficulties for their implementation, which needs to be at least considered. This review paper intends to quickly summarize the efforts performed to produce these artificial enzymes bearing several active centers, putting more emphasis on the discussion of their possibilities and some practical problems that can be envisioned and should be considered in future developments. ## Enzymes bearing several active centers In this section, we will list different examples where enzymes bearing two active centers are created. ## Natural enzymes We have been unable to find reports describing natural enzymes bearing two independently active centers except when some natural fusion enzymes are formed, but these will be treated in the next section. An exception to this fact is the enzymes exhibiting activity promiscuity. Substrate promiscuity is related to enzyme specificity, and it is not a surprise that an enzyme can recognize many different substrates, since many enzymes have low specificity (an outstanding example are lipases, which are able to recognize many different substrates) or can catalyze diverse-related reactions using the same catalytic center that is used for the physiological reaction, as happens in ester hydrolysis, esterification, transesterification and so on [bib_ref] Industrial applications of microbial lipases, Hasan [/bib_ref] [bib_ref] Lipases for biotechnology, Jaeger [/bib_ref] [bib_ref] Microbial lipases form versatile tools for biotechnology, Jaeger [/bib_ref]. These are enzymes with a broad specificity and are able to catalyze very diverse reactions. However, here we refer to a promiscuous activity, that may be defined as the capacity of the enzyme to catalyze a reaction far from the physiological activity of the enzyme, that is, with different chemo-selectivity. An example may be a hydrolase able to produce carbon-carbon bonds or exhibiting an oxidative activity. This is the real enzyme promiscuity, and it is postulated that it has been somehow related to the evolution of organisms, as first step before gene diversification to obtain new enzyme activities [bib_ref] The "evolvability" of promiscuous protein functions, Aharoni [/bib_ref] [bib_ref] Catalytic promiscuity and the evolution of new enzymatic activities, O&apos;brien [/bib_ref] [bib_ref] Enzyme promiscuity: Evolutionary and mechanistic aspects, Khersonsky [/bib_ref]. These promiscuous activities may be of interest when there are no alternative natural enzymes able to perform the reaction [bib_ref] Lipase promiscuity and its biochemical applications, Kapoor [/bib_ref] [bib_ref] Kinetic model of the enzymatic Michael addition for synthesis of mitomycin analogs..., Zhang [/bib_ref] [bib_ref] Advances in Biocatalytic Promiscuity: Hydrolase-Catalyzed Reactions for Nonconventional Transformations, Lõpez-Iglesias [/bib_ref] [bib_ref] Catalytic promiscuity in biocatalysis: Using old enzymes to form new bonds and..., Bornscheuer [/bib_ref] [bib_ref] Enzyme promiscuity: Mechanism and applications, Hult [/bib_ref] [bib_ref] Hydrolases: Catalytically promiscuous enzymes for non-conventional reactions in organic synthesis, Busto [/bib_ref] [bib_ref] Biocatalytic promiscuity, Humble [/bib_ref]. In many examples, it has been shown that this secondary catalytic activity requires a folded enzyme structure (that is, it is not a consequence of the catalytic activity of individual amino acids) but that did not involve the active center of the enzyme (as the activity remains after blocking the active center of the enzyme with irreversible inhibitors) [bib_ref] Tuning lipase B from Candida antarctica C-C bond promiscuous activity by immobilization..., Izquierdo [/bib_ref] [bib_ref] Alternate-site enzyme promiscuity, Taglieber [/bib_ref]. This can suggest that the active center responsible for this activity may be placed in a different position, perhaps in a different pocket of the enzyme surface . As the standard enzyme activity, this promiscuous activity may be modulated by enzyme immobilization [bib_ref] Tuning lipase B from Candida antarctica C-C bond promiscuous activity by immobilization..., Izquierdo [/bib_ref]. However, the catalytic activity values are usually moderate, and obviously, the main active center and this promiscuous activity may be utilized to catalyze cascade reactions only randomly [bib_ref] Enzyme-Catalyzed Asymmetric Domino Thia-Michael/Aldol Condensation Using Pepsin, Xiang [/bib_ref]. Nevertheless, we wanted to mention this possibility as an example of natural enzymes bearing two active centers. ## Enzyme main active center Secondary pocket with promiscuous activity . Enzymes having promiscuous activities not related to the main active center. ## Fusion enzymes The building of enzymes bearing a domain has been a traditional strategy to facilitate enzyme purification, producing chimeric enzymes using very simple and small tags, such as poly-His (using immobilize metal chelate columns) [bib_ref] Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports, Armisén [/bib_ref] [bib_ref] Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using..., Kumar [/bib_ref] or poly-Lys tags (to purify the enzyme using anionic exchanges), taking advantage of the usual low isoelectric point of enzymes [bib_ref] Evaluation of employing poly-lysine tags versus poly-histidine tags for purification and characterization..., Wijekoon [/bib_ref] to large domains that are designed to introduce affinity moieties, such as cellulose binding domain or choline binding domain [bib_ref] Versatility of choline-binding domain, García [/bib_ref] [bib_ref] Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial..., Terpe [/bib_ref]. These affinity domains may also be used as a single step immobilization purification of enzymes [bib_ref] Positively charged mini-protein Z basic2 as a highly efficient silica binding module:..., Bolivar [/bib_ref] [bib_ref] Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic..., Bolivar [/bib_ref] [bib_ref] Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger, Kweon [/bib_ref] [bib_ref] Oriented immobilization of enzymes made fit for applied biocatalysis: Non-covalent attachment to..., Wiesbauer [/bib_ref] [bib_ref] Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors, Nilsson [/bib_ref] [bib_ref] Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes, Richins [/bib_ref] [bib_ref] Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts, Barbosa [/bib_ref] [bib_ref] Functionalization of gold surfaces for specific and reversible attachment of a fused..., Madoz [/bib_ref] [bib_ref] Immobilization and single-step purification of fusion proteins using DEAE-cellulose, Sanchez-Puelles [/bib_ref] [bib_ref] One-step immobilization-purification of enzymes by carbohydratebinding module family 56 tag fusion, Qin [/bib_ref] [bib_ref] Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization..., Moldes [/bib_ref]. However, nature has shown us that this may be also used to produce enzymes bearing several catalytic domains by fusing the domains of different enzymes. One example of this is the covalent fusion of soluble P450 and cytochrome P450 reductase enzymes from Bacillus megaterium to produce the flavocytochrome P450 BM3 system, and to achieve a highly efficient electron transport system for oxygenation of fatty acids and related molecules [bib_ref] Cytochrome P450-redox partner fusion enzymes, Munro [/bib_ref]. This gene fusion seems to be a natural evolutionary event due to fusion of adjacent genes [bib_ref] Enzyme evolution: Generation of a bifunctional enzyme by fusion of adjacent genes, Yourno [/bib_ref] [bib_ref] Prediction of protein interactions: Metabolic enzymes are frequently involved in gene fusion, Tsoka [/bib_ref]. Considering the advantages of having a single molecule bearing two desired catalytic activities, researchers have tried to imitate nature and produce fusion enzymes for a long time [fig_ref] Figure 2: Design of artificially fused enzymes [/fig_ref]. These fusion enzymes have been used in biomedicine [bib_ref] Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic..., Kan [/bib_ref] , in vivo to produce target metabolites via fermentation [bib_ref] Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of..., Albertsen [/bib_ref] [bib_ref] Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via..., Lee [/bib_ref] [bib_ref] Improved production of arachidonic acid by combined pathway engineering and synthetic enzyme..., Liu [/bib_ref] [bib_ref] Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae, Rabeharindranto [/bib_ref] [bib_ref] Enzyme fusion removes competition for geranylgeranyl diphosphate in carotenogenesis, Camagna [/bib_ref] [bib_ref] Construction of a fusion enzyme for astaxanthin formation and its characterisation in..., Nogueira [/bib_ref] or as isolated bifunctional enzymes to catalyze cascade reactions [bib_ref] Construction and function of fusion enzymes of the human cytochrome P450scc system, Harikrishna [/bib_ref] [bib_ref] Do Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate..., Seo [/bib_ref] [bib_ref] Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion, Bulow [/bib_ref] [bib_ref] Heterologous gene expression in Aspergillus niger: A glucoamylase-porcine pancreatic prophospholipase A2 fusion..., Roberts [/bib_ref] [bib_ref] Enhancing the enzymatic hydrolysis efficiency of lignocellulose assisted by artificial fusion enzyme..., Du [/bib_ref] [bib_ref] Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for..., Liao [/bib_ref] [bib_ref] Production of indigo through the use of a dual-function substrate and a..., Fabara [/bib_ref] [bib_ref] What to sacrifice? Fusions of cofactor regenerating enzymes with Baeyer-Villiger monooxygenases and..., Mourelle-Insua [/bib_ref] [bib_ref] Biochemical characterization of a bifunctional enzyme constructed by the fusion of a..., Baklouti [/bib_ref] [bib_ref] Preparation of a soluble bifunctional enzyme by gene fusion, Bülow [/bib_ref]. The selection of appropriate linkers between the involved enzymes is a critical point in the design of these fusion proteins, to permit a proper folding of both fused enzymes [bib_ref] Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers, Lu [/bib_ref]. In some instances, the researchers fused more than two enzymes. ## Enzyme 1 ## Enzyme 2 ## Linker Fused enzyme 1-2 Genetic fusion with a suitable linker These fusion proteins can be produced genetically, fusing coding open reading frames, or the connection of the proteins may be performed in a posttranslational process. This interesting topic has been the subject of many interesting reviews [bib_ref] Bringing functions together with fusion enzymes-from nature's inventions to biotechnological applications, Elleuche [/bib_ref] [bib_ref] Enzyme fusions in biocatalysis: Coupling reactions by pairing enzymes, Aalbers [/bib_ref] [bib_ref] Microbial lipolytic fusion enzymes: Current state and future perspectives, Gudiukaite [/bib_ref] , therefore, we are not going to extend further in this matter. ## Modification of enzymes with non-biological catalysts The combination of enzyme and metal catalysis is highly interesting and can open new synthetic possibilities. Unfortunately, in many instances the combined use of both catalysts lead to mutual inactivation, making some compartmentalization necessary to prevent this negative effect [bib_ref] New reaction pathways by integrating chemo-and biocatalysis, Bering [/bib_ref]. However, another alternative is to prepare enzymes bearing both catalytic activities, via the modification of the enzymes with a compound bearing the desired catalytic activity. This has been recently reviewed [bib_ref] Artificial enzymes with multiple active sites, Palomo [/bib_ref]. Here, we will just give some examples to show the potential. For example, glucose oxidase was coated with hemin (a peroxidase-mimetic catalytic polymer) via a flexible polymeric scaffold through coordination to their imidazole groups. This spatial distribution allows the enzyme to catalyze its reaction first and then hemin catalyst acts. This was used to build nanoreactors able to degrade organic aromatic compounds using glucose as the only fuel [bib_ref] Nanoconfined (bio)catalysts as efficient glucose-responsive nanoreactors, Rodriguez-Abetxuko [/bib_ref]. Another strategy consists of using the enzyme as a scaffold to get a metal nanoparticle attached to the enzyme [fig_ref] Figure 3: Preparation of an enzyme bearing its biological center and a nanoparticle [/fig_ref]. In fact, the use of different organisms to produce transition metal nanoparticles in a greener way has been proposed by some authors, in vivo or in vitro [bib_ref] Synthesis of metallic nanoparticles using plant extracts, Mittal [/bib_ref] [bib_ref] Synthesis in plants and plant extracts of silver nanoparticles with potent antimicrobial..., Mashwani [/bib_ref] [bib_ref] Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold..., Vetchinkina [/bib_ref] [bib_ref] Marine microorganisms for synthesis of metallic nanoparticles and their biomedical applications, Patil [/bib_ref] [bib_ref] Green synthesis and characterization of silver nanoparticles by Allium cepa L. to..., Sharma [/bib_ref] [bib_ref] Green synthesis of silver nanoparticles using water extract of Salvia leriifolia: Antibacterial..., Baghayeri [/bib_ref] [bib_ref] Biosynthesis of palladium nanoparticles using Shewanella loihica PV-4 for excellent catalytic reduction..., Wang [/bib_ref] [bib_ref] Synthesis of nanoparticles by microorganisms and their application in enhancing microbiological reaction..., Zhang [/bib_ref] [bib_ref] Nanoparticles biosynthesized by fungi and yeast: A review of their preparation, properties,..., Boroumand Moghaddam [/bib_ref] [bib_ref] Ag and Au-Ag nanoparticles using edible mushroom extract, Philip [/bib_ref]. The production of nanoparticles employing enzymes as inductors of the metal nanoparticle formation in aqueous media is a well-known possibility [bib_ref] Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold..., Vetchinkina [/bib_ref] [bib_ref] Growing metal nanoparticles by enzymes, Willner [/bib_ref] [bib_ref] Nanobiohybrids: A new concept for metal nanoparticles synthesis, Palomo [/bib_ref]. Thus, it is possible to build enzymes bearing metal nanoparticles to get multifunctional hybrid-enzymes. One example of this may be the use of an alcohol oxidase to produce an in situ nanoparticle, used as an amperometric alcohol biosensor [bib_ref] Alcohol oxidase protein mediated in-situ synthesized and stabilized gold nanoparticles for developing..., Chinnadayyala [/bib_ref]. There are many other interesting examples of this kind of hybrid biocatalyst [bib_ref] Design of enzyme-metal hybrid catalysts for organic synthesis, Li [/bib_ref] [bib_ref] Synthesis of highly active enzyme-metal nanohybrids and uncovering the design rules, Cheng [/bib_ref] [bib_ref] Construction of chemoenzymatic cascade reactions for bridging chemocatalysis and biocatalysis: Principles, strategies..., Liu [/bib_ref] [bib_ref] Optimisation of catalysts coupling in multi-catalytic hybrid materials: Perspectives for the next..., Heuson [/bib_ref]. ## Metal nucleation pocket Metallic salt solution Enzyme bearing the biological active center and a catalytic nanoparticle In some instances, this strategy may also solve the problem of the biocatalyst solubility. For example, lipase B from Candida antarctica was mixed with different metal salts (Pd(OAc) 2 , Na 2 PdCl 4 , AgNO 3 and HAuCl 4 ), to produce a solid precipitate that could be used as an ex novo heterogeneous biocatalyst [bib_ref] Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications..., Filice [/bib_ref]. The enzyme activity recovery depended on the metal. This biocatalyst was utilized in the cascade reaction (enzymatic hydrolysis plus metal catalyzed reduction) for the transformation of p-nitrophenyl butyrate to p-aminophenol [bib_ref] Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications..., Filice [/bib_ref]. One obvious alternative to this strategy is to utilize the enzyme immobilized in a nanoparticle containing the metal [bib_ref] Assembly of nano-biocatalyst for the tandem hydrolysis and reduction of p-nitrophenol esters, Barros [/bib_ref]. While the first strategy utilizes a green method to produce the aggregate in one step, the second permits better control of the enzyme-support interactions, which, as discussed in the introduction section, may permit a better final enzyme stability/activity. Another example of the preparation of a metal/enzyme precipitate is the production of an oxidase precipitate hosting small spherical palladium nanoparticles that present catalytic competence for both the biocyclization as well as the C-C bond-forming cross coupling [bib_ref] Arylative allenol cyclization via sequential one-pot enzyme & palladium catalysis, Naapuri [/bib_ref]. ## Metallic salt solution Enzyme-metal precipitate with biological and metallic catalytic activities These strategies may present some problems, because the metal can still be dissolved under certain conditions affecting the enzyme activity and also decreasing the metal activity. The insertion of an organometallic catalyst onto a specific point of the enzyme is a more elegant way to reach the desired goal [fig_ref] Figure 6: Preparation of enzyme site-directed modified with a metal catalyst [/fig_ref]. In one interesting example, the lipase from Geobacillus thermocatenulatus was utilized. This is a peculiar lipase presenting a double lid [bib_ref] Activation of bacterial thermo alkalophilic lipases is spurred by dramatic structural rearrangements, Carrasco-López [/bib_ref]. They optimized the solid-phase modification of the active center of the enzyme [bib_ref] Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity, Filice [/bib_ref] , and later on, this protocol was extrapolated to the modification of the position 196 (where a Cys had been genetically introduced), located in the external face of the main lid, with a thiol reactive-organometallic complex. The position where the Cys was introduced was justified by the authors because the introduction on this position of a peptide had previously permitted a strong modulation of the enzyme selectivity, revealing their importance [bib_ref] Effect of site-specific peptide-tag labeling on the biocatalytic properties of thermoalkalophilic lipase..., Romero [/bib_ref]. Very interestingly, the authors showed the great impact of the immobilization strategy on the expressed activity of the introduced organometallic catalyst [bib_ref] Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity, Filice [/bib_ref]. This was explained by the necessity of having the open form of the lipase to leave accessible the new catalytic groups. The new immobilized hybrid enzyme was utilized in a cascade reaction to produce aminoarene from a nitroarene ester [bib_ref] Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity, Filice [/bib_ref]. Another strategy to create artificial enzymes is the introduction of noncanonical amino acids with catalytic activity in proteins to give some catalytic activities via genetic modification [bib_ref] Directed evolution of a designer enzyme featuring an unnatural catalytic amino acid, Mayer [/bib_ref] [bib_ref] Design and evolution of an enzyme with a non-canonical organocatalytic mechanism, Burke [/bib_ref] [bib_ref] Expanding the enzyme universe with genetically encoded unnatural amino acids, Drienovská [/bib_ref]. When combining this and the previously described tools, in a further effort, a protein may be transformed in an enzyme bearing two artificial active centers. The researchers introduced (on the lactococcal multidrug resistance regulator) a genetically encoded unnatural p-aminophenylalanine residue. This group is able to activate an enol via iminium ion formation. They also introduced a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and supplies it to one preferred prochiral face of the activated enal) [bib_ref] Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological..., Zhou [/bib_ref]. That way the final hybrid biocatalyst was able to act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in a catalyzed Michael addition reaction. Using a similar double modification, a biocatalyst able to catalyze the tandem Michael addition/enantioselective protonation highly enantioselective reaction was also prepared [bib_ref] Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological..., Zhou [/bib_ref]. ## Genetic introduction of a cys in a proper enzyme pocket Directed chemical modification with a -thiol metal chelating agent Enzyme bearing biological and metal catalytic activities ## Design of enzymes bearing an ex novo biological active center (plurizymes) The combined use of enzyme modelling, dynamic simulation and genetic tools have enabled the design of the so-called plurizymes by the research group directed by Prof. Ferrer [bib_ref] Rational engineering of multiple active sites in an ester hydrolase, Santiago [/bib_ref]. This novel strategy consists of the search (using enzyme modeling and dynamic simulation) on the surface of an enzyme for pockets that can be suitable to create a new, ex novo, human-designed biological active center (via a minimum of site directed mutations). This ex novo artificial active center will add its catalytic activities to that of the initial active center. That way, an enzyme bearing two biological (one natural and one artificial ones) will be created [bib_ref] Rational engineering of multiple active sites in an ester hydrolase, Santiago [/bib_ref] [fig_ref] Figure 7: Design and optimization of plurizymes [/fig_ref]. The first approach was the introduction of a second serine hydrolase active center (including the whole catalytic triad) in a serine ester hydrolase obtained by metagenomics approaches [bib_ref] Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): A..., Martínez-Martínez [/bib_ref] and previously identified as the ester hydrolase, with the broadest specificity among a total of 147 esterases assayed [bib_ref] Determinants and prediction of esterase substrate promiscuity patterns, Martínez-Martínez [/bib_ref]. This enzyme was studied to identify likely pockets where the new catalytic triad may be accommodated and the substrate may be adsorbed (using tripropionin as a model substrate), and employing Protein Energy Landscape Exploration (PELE) software (because it enables mapping ligand diffusion and binding) [bib_ref] Determinants and prediction of esterase substrate promiscuity patterns, Martínez-Martínez [/bib_ref] [bib_ref] Software News and Updates Gabedit-A Graphical User Interface for Computational Chemistry Softwares, Allouche [/bib_ref] [bib_ref] Substrate diffusion and oxidation in GMC oxidoreductases: An experimental and computational study..., Hernández-Ortega [/bib_ref] [bib_ref] A benchmark exercise using unpublished data from pharma, Carlson [/bib_ref] [bib_ref] Computer-aided laccase engineering: Toward biological oxidation of arylamines, Santiago [/bib_ref]. A pocket on the enzyme surface containing a Ser (residue 211) was identified, and Asp and His residues were first computationally and later experimentally added (Glu25Asp and Leu214His) to generate a catalytic triad. Special care was taken in the distances between the residues and substrate accommodation [bib_ref] Rational engineering of multiple active sites in an ester hydrolase, Santiago [/bib_ref]. The near Gly207, Tyr208 and Phe209 groups generate a likely oxyanion hole. The natural catalytic Ser of the native enzyme was then mutated (Ser161Ala) to eliminate the native enzyme activity and ensure the functionality of the new active center. The authors found activity of the new active center versus 24 substrates, obviously with different specificities and activities when compared to the native active center. This catalytic activity was dependent on the two mutations introduced to generate the catalytic triad as well as of the natural Ser211. Then, the authors generated an enzyme bearing the two intact catalytic centers. The specific activity of the double active center enzyme for all accepted esters was lower than that observed for the native enzyme, but higher than that for the enzyme bearing only the artificial active center [bib_ref] Rational engineering of multiple active sites in an ester hydrolase, Santiago [/bib_ref]. The enzyme specificity was not enlarged, as the scaffold enzyme was already one esterase bearing a very broad specificity. However, the enzyme bearing the two active centers gave an altered substrate/activity (giving a sigmoidal curve) and pH/activity (giving a narrower peak) curves. This is the only example of a design of plurizymes in the literature to date, and it may be considered a proof-of-concept using relatively simple active centers to facilitate the success. However, it is expected that many other examples may follow this model, perhaps plurizymes, which not only show two different active centers, but also with two different catalytic activities. In a further step, the model plurizyme features were improved by site-directed mutagenesis [bib_ref] Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic..., Alonso [/bib_ref]. The objective was to achieve better spatial configuration of the active center, and they were able to produce an improved plurizyme with increased catalytic activity (even by 74-folds), an increased enantiospecificity (by over 1000-fold) and an increased temperature of 20 - C, at which point the enzyme retained more than 80% of its optimal activity. It also expanded the substrate scope, as the resulting plurizyme presented activity versus some substrates that were not recognized by the original esterase [bib_ref] Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic..., Alonso [/bib_ref]. ## Selection of a suitable ## Design plurizymes that are site-directed modified with organometallic catalysts The modified plurizymes described in the previous section were also the first (and only at present) examples of a new strategy to obtain plurizymes bearing a biological and a metal-organic catalytic center [bib_ref] Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic..., Alonso [/bib_ref] [fig_ref] Figure 8: Design and modified plurizymes [/fig_ref]. The researchers designed an irreversible serine hydrolase inhibitor attached to an organometallic catalytic complex. To reach this goal, 3 -hydroxy-2,2 -bipyridin-3-yl methyl hexylphosphonate, coupled to a transitionmetal-chelating moiety (the bipyridine ligand) was synthesized. The use of an excess of the inhibitor permitted blocking of both native (Ser161) and artificial (Ser211) catalytic groups. The double-blocked enzyme, after incubation with Cu(NO 3 ) 2, exhibited a synergy in the oxidation of catechol, as the double-modified enzymes were more active than both individually modified enzymes. The authors postulated that the proximity of the two organometallics facilitated the intramolecular electron transfer. In this way, one advantage of using plurizymes was found [bib_ref] Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic..., Alonso [/bib_ref]. However, this composite still has only one catalytic activity bearing two active centers. To have a plurizyme bearing two different activities, the researchers utilized a different enzyme specificity for the different substrates, and therefore, for the inhibitor. They found that the native active center of the plurizyme presented a much higher affinity for the inhibitor than the artificial one. In that way, using almost a stoichiometric amount of inhibitor, they were able to selectively inhibit the native active site while leaving the artificially introduced one unaltered. This new mono-modified plurizyme presented two different catalytic activities and could be utilized as single catalyst of different one pot cascade reactions. For example, it was used to transform 1-naphthyl acetate into 1,4-naphthoquinone (with a conversion near to 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (with a conversion next to 85% and an enantiomeric excess of 99.9%) [bib_ref] Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic..., Alonso [/bib_ref]. The versatility of this strategy to introduce different active centers, benefitting from the environment generated by the active enter pocket, may open unlimited opportunities to prepare catalysts able to catalyze the most complex processes under the milder and most selective conditions. We foresee a great development in the area of plurizymes and modified plurizymes in the upcoming future. ## Practical problems of enzymes bearing several active centers The developments to generate artificial enzymes bearing several active centers that have been discussed above are academically very relevant and may open the door to new and unexpected advances in the design of bioprocesses that some years ago seemed only to be a dream. Moreover, some developments are so recent that the full impact that they may have are still unknown, similar to the modified plurizymes. However, we can foresee some problems for the application of these artificial enzymes bearing several active centers. For example, one critical step in the optimization of multi-enzymatic processes is the optimization of the ratio between the activities of the involved catalytic entities [bib_ref] Multi-enzymatic cascade reactions: Overview and perspectives, Ricca [/bib_ref] [bib_ref] Cell-free metabolic engineering: Production of chemicals by minimized reaction cascades, Guterl [/bib_ref] [bib_ref] Multienzyme cascade reactions-Status and recent advances, Sperl [/bib_ref] [bib_ref] Multi-enzymatic synthesis, Lopez-Gallego [/bib_ref] [bib_ref] Wiring step-wise reactions with immobilized multi-enzyme systems, Velasco-Lozano [/bib_ref]. The problem has more or less importance depending on the strategy utilized to have several active centers. Using fusion multi-enzyme composites, it is theoretically possible to add more or fewer enzyme-units to the fusion target enzyme to have the desired activity ratio, or, more simply, to add the required amount of the individual enzyme, which is less active. However, if using the alternatives involving just one enzyme structure where a new active center is created, the optimization may be more difficult. If the activity that needs to be used in excess is that belonging to the native active center of the enzyme, it may be possible to use a mixture of the natural enzyme and the artificial one to get the desired native/artificial activities ratio (assuming that the properties of the natural and the modified enzymes are similar, that may be untrue in many instances). If the activity that needs to be reinforced is that from the artificial active center, to get compensated activities ratios may require using some multi-activity enzymes having the native enzymatic catalytic center inactivated and only bearing the artificial active center. If the artificial activity is less active and it is the second in the chain, it may produce the accumulation of its substrate. If this substrate is unstable (e.g., tends to isomerize or racemize, or it is easily oxidable), the result of the process will not be the desired one, as a percentage of the intermediate product will be destroyed [bib_ref] The coimmobilization of d-amino acid oxidase and catalase enables the quantitative transformation..., Fernández-Lafuente [/bib_ref] [bib_ref] Immobilization of genetically-modified D-amino acid oxidase and catalase on carbon nanotubes to..., Li [/bib_ref] [bib_ref] D-Amino acid oxidase and catalase of detergent permeabilized Rhodotorula gracilis cells and..., Upadhya [/bib_ref] [bib_ref] Characterization and application of d-amino acid oxidase and catalase within permeabilized Pichia..., Tan [/bib_ref] [bib_ref] Enzyme production of d-gluconic acid and glucose oxidase: Successful tales of cascade..., Kornecki [/bib_ref] [bib_ref] Hydrogen peroxide in biocatalysis. A dangerous liaison, Hernandez [/bib_ref] [bib_ref] Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade: A..., Mateo [/bib_ref]. Another likely problem may rise if the stability of one of the activities is much lower than the stability of the other components of the chain. This way, this weak component will mark the stability of the whole composite, causing the same problem that arises when using coimmobilized native enzymes . Using coimmobilized enzymes, some solutions have been recently proposed for these dissimilar stabilities of the different enzyme components, designing strategies that permit the release of the least enzyme component after its inactivation and the immobilization of a fresh enzyme batch [bib_ref] Enzyme co-immobilization: Always the biocatalyst designers' choice . . . or not?, Arana-Peña [/bib_ref] [bib_ref] The combination of covalent and ionic exchange immobilizations enables the coimmobilization on..., Arana-Peña [/bib_ref] [bib_ref] Chemical amination of immobilized enzymes for enzyme coimmobilization: Reuse of the most..., Carballares [/bib_ref] [bib_ref] Coimmobilization of lipases exhibiting three very different stability ranges. Reuse of the..., Carballares [/bib_ref] [bib_ref] Preparation of a six-enzyme multilayer combi-biocatalyst: Reuse of the most stable enzymes..., Carballares [/bib_ref] [bib_ref] Coimmobilization of enzymes in bilayers using PEI as a glue to reuse..., Zaak [/bib_ref] [bib_ref] Advantages of supports activated with divinyl sulfone in enzyme coimmobilization: Possibility of..., Morellon-Sterling [/bib_ref] [bib_ref] New applications of glyoxyl-octyl agarose in lipases co-immobilization: Strategies to reuse the..., Arana-Peña [/bib_ref]. This solution is not valid using enzymes bearing multiple activities; the only solutions will be as described above to get a balanced ratio between the involved activities. One exception may be (as we have not found any paper concerning this possibility, this may be just a hypothesis) the case where we use an enzyme bearing a metallic catalyst together with the biological active center. If the problem is in the inactivation of the metal itself and it is not caused by some undesired conformational change of the enzyme structure in the area where the metal is adsorbed, its activity could be recovered. In this case, it might be possible to release the inactivated metal and reload fresh metal batches following a similar protocol to the employed in the preparation of the original artificial organometallic-enzyme complex. If the cause of metal inactivation is a conformational change affecting the area where the metal is assembled and not to the enzyme active center, the solutions will require adding enzyme containing only the metallic active center. (obviously if the accumulation of the substrate of the metal produces some undesired effect as described above). In this way, although conceptually and academically, the design of enzymes bearing several active centers is of indubitable interest, and their practical implementation should consider how to solve these likely drawbacks, and some other problems that might have been undetected to date. # Conclusions The design of enzymes bearing a unique structure and several catalytic activities is of great interest. Starting with natural evolution, similar to fusion enzymes or enzymes bearing a second promiscuous active center, nowadays the tendency is to add catalytic active centers whose activities are quite far from the biological ones, permitting the coupling of biological and metal catalysis to perform the most complex, selective and environmentally friendly cascade reactions. In many instances, as in the development of plurizymes and modified plurizymes, they were only possible by the imagination of the involved researchers and the availability of accurate and powerful enough tools to perform enzyme modelling and design, as well as organic chemistry (to build the suicide inhibitor attached to the organometallic catalysts). These novel tools should be a topic to be strongly developed in the near future, as they may permit the relatively simple coupling of biological and metal catalysis, but to date these tools are clearly under-exploited. This huge potential should not cause researchers to forget some practical problems, such as the necessity of use balanced activities of the involved enzymes to maximize the catalytic potential of the new multiple activity enzymes, or the possibility of very different operational stability of their different elements. While immobilization is usually considered a requisite in most of the industrial applications of enzymes (and most of the enzyme is the result of a sophisticated post-production enzyme chemical modification that can be benefited of the use of solid phase), scarce efforts have been directed to the immobilization of some of these multiple activity enzymes, with the exception of enzymes/metal precipitated and one example of immobilization of an enzyme site directed modified with an organometallic catalyst. It is also likely that this gap may be covered in the upcoming future. Thus, the application of cascade reactions may be enlarged by the use of biological and metal catalysis but still benefit from the selectivity, reactivity and specificity given by the biological environment. Although academically they are very relevant, the practical advantages of using simultaneously individual natural enzymes and artificial enzyme catalyst bearing just one active center in a cascade reaction versus having both activities in the same unique structure have not been proved in most cases. One exception is the synergy found by the proximity of two organometallic complexes in a modified plurizymes. This synergy between active centers may become an advantage to consider in the design of multi activity enzymes. The future should drive to never stop dreaming of multiple activity enzymes where these advantages may be clearly established, as now we are in the starting of the development of some of the new technologies (e.g., plurizymes). [CrossRef] 44. [fig] Figure 2: Design of artificially fused enzymes. [/fig] [fig] Figure 3: Preparation of an enzyme bearing its biological center and a nanoparticle. [/fig] [fig] Figure 4, Figure 5: Preparation of metal-enzyme aggregates. Preparation of enzymes immobilized in metal catalyst nanoparticle. [/fig] [fig] Figure 6: Preparation of enzyme site-directed modified with a metal catalyst. [/fig] [fig] Figure 7: Design and optimization of plurizymes. [/fig] [fig] Figure 8: Design and modified plurizymes; single or multiple catalytic activities. [/fig] [fig] Author: Contributions: D.C. and R.M.-S. performed the initial bibliographic search and prepare figures, R.F.-L. supervised the writing and write the final version of the manusvript. All paper performed a final revision of the paper. All authors have read and agreed to the published version of the manuscript. Funding: We gratefully recognize the financial support from the Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R) and the Agencia Estatal de Investigación (PID2021-122398OB-I00). R.M.-S. thanks the Ministerio de Educacion-Spanish Government for an FPU fellowship, D.C. thanks the Ministerio de Ciencia e Innovación-Spanish Government for an FPI. [/fig]

The use of statins was associated with reduced COVID-19 mortality: a systematic review and meta-analysis Background: Statins are widely used to treat people with metabolic and cardiovascular disorders. The effect of statins on coronavirus disease 2019 (COVID-19) is unclear. To investigate the association between statins and COVID-19 outcomes and, if possible, identify the subgroup population that benefits most from statin use. Materials and methods: A systematic review and meta-analysis of published studies that included statin users and described COVID-19 outcomes through 10 November 2020. This study used the generic inverse variance method to perform meta-analyses with random-effects modelling. The main outcomes were evaluation of the need for invasive mechanical ventilator (IMV) support, the need for intensive care unit (ICU) care and death. All outcomes were measured as dichotomous variables. Results: A total of 28 observational studies, covering data from 63,537 individuals with COVID-19, were included. The use of statins was significantly associated with decreased mortality (odds ratio [OR] ¼ 0.71, 95% confidence interval [CI]: 0.55-0.92, I 2 ¼72%) and the need for IMV (OR ¼ 0.81, 95% CI: 0.69-0.95, I 2 ¼0%) but was not linked to the need for ICU care (OR ¼ 0.91, 95% CI: 0.55-1.51, I 2 ¼66%). Subgroup analysis further identified five types of studies in which statin users had even lower odds of death. Conclusions: The use of statins was significantly associated with a reduced need for IMV and decreased mortality among individuals with COVID-19. Statins may not need to be discontinued because of concern for COVID-19 on admission. Further randomized controlled trial (RCTs) are needed to clarify the causal effect between statin use and severe COVID-19 outcomes.KEY MESSAGESParticipants in five types of studies were shown to have even lower odds of death when taking statins. The use of statins was significantly associated with a reduced need for invasive mechanical ventilation and decreased all-cause mortality among individuals with COVID-19. However, statin use did not prevent participants from needing care in the intensive care unit. The results justify performing randomized controlled trials (RCTs) to validate the benefits of statins on COVID-19 outcomes. # Introduction Coronavirus disease 2019 (COVID-19) has been a tremendous threat to human life since the pandemic began. People at high risk of unfavourable outcomes caused by COVID-19 include elderly persons and individuals who have chronic metabolic and cardiovascular comorbidities. Statins are a class of medications that are widely used to manage metabolic syndrome and cardiovascular diseases. A nationwide report showed that up to 30% of the population aged !45 years took statins. Whether the use of statins will affect the clinical course and prognosis of COVID-19 is gaining increasing attention. Statins are primarily used to lower serum lipids by inhibiting HMG-CoA reductase. In addition, statins are also known to have pleiotropic effects, includinganti-inflammation, (2) immunomodulation, (3) upregulation of angiotensin-converting enzyme 2 (ACE2) receptor expression, (4) antithrombosis and (5) antioxidation. Some of the effects may have complicated interactions with COVID-19. For example, ACE2 is the main entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Chronic use of statins may theoretically increase susceptibility to COVID-19. On the other hand, ACE2 mediated conversion of angiotensin II to angiotensin (1-7) peptide, which can protect the lung from acute injury. The overall influence of statin use on COVID-19 remains unclear. An increasing number of researchers are focussing on the relationship between statins and COVID-19. Currently, these studies are all observational, and there are no published randomized controlled trials (RCTs). To date, most studies agree that statins do not have to be discontinued after the onset of COVID-19. However, there is still no concrete conclusion about the impact of statins on the prognosis of COVID- This study aims to explore the relationship between statins and COVID-19. Through a comprehensive systematic review and meta-analysis, we expect to answer two clinical questions: (1) will the use of statins lead to a favourable/poor outcome of COVID- can statins decrease the mortality of COVID-19 in a subgroup of statin users? The study results will provide valuable information to health care workers and guide the choice of potential participants in future RCTs. # Materials and methods ## Search strategy This study was a systematic review and meta-analysis conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations . We searched the PubMed, Cochrane Library, Embase and MEDLINE Online databases for relevant articles published through 10 November 2020, using the keywords and related MeSH terms as follows: "COVID-19", "statin' and "HMG-CoA reductase". There was no restriction on language or publication status. The reference lists of eligible studies were also manually searched to identify any additional relevant studies. Detailed search strategies are described in. ## Selection criteria The inclusion criteria for this study are described as follows in the order of population, intervention, comparator, outcome, study design (PICOS) formulation. The population were individuals with positive/confirmed COVID-19; the intervention was the use of statin before or during the hospital course of COVID-19; the comparators were those who did not take statin before or during COVID-19. This study set up three outcomes to evaluate the relationship between statin use and COVID-19, including intensive care unit (ICU) care, use of an invasive mechanical ventilator (IMV) and death. Any study that described at least one of the outcomes was included for assessment and analysis. Eligible study designs included RCT, cohort, clinical trial, case-cohort and cross-over design. The following types of articles were excluded, including review articles, hypothesis, case reports, articles in a non-English language, articles focussing on paediatric populations (17 years of age or younger), articles with a Newcastle-Ottawa scale (NOS) score of 6 or below, articles providing inadequate information and articles not relevant to the study goal. Two investigators (TCP and PLT) independently performed a systematic review using the same criteria and included studies on the basis of agreement. Upon disagreement, a third investigator (PCL) joined and helped make the final decision. ## Data extraction Eligible studies were first deduplicated by EndNote X9. Then, two investigators (KSW and PLT) independently extracted data from the included studies using an established data collection form. Collected variables included the first author's name, publication year, study country, study design, sample size, study period, demographics of participants, follow-up duration, diagnostic method of COVID-19, study quality and outcomes. We also contacted corresponding authors to gather missing data when needed. Both raw data and results presented as relative risk/odds ratio (OR)/hazard ratio were included. If a study provided unadjusted and adjusted results, we extracted the adjusted results. The case fatality rate (CFR) of each study was calculated using the number of deceased individuals divided by the number of all individuals. Any discrepancies in data extraction or quality assessment were discussed with a third investigator (TCP) to reach a final agreement. ## Quality and risk of bias assessments This study used the Cochrane risk of bias tooland NoSto appraise the quality of RCTs and observational studies. The NoS consists of eight items covering three broad perspectives: The selection of the study groups, the comparability of the groups and the ascertainment of either the exposure or outcome of interest. The total maximum score of these three perspectives is nine. A study that scores equal to or higher than seven is considered high-quality research. Besides, a funnel plot was used to evaluate the existence of publication bias. ## Data synthesis and statistical analysis This study used the generic inverse variance method to perform meta-analysis since some of the enrolled studies reported results with an inverse design. We conservatively chose random effect modelling for analysis since differences in the study population and study design between studies were expected. If an article presented both unadjusted and adjusted results, we retrieved the adjusted results for meta-analysis. This study used the odds ratio to present the overall effect estimates in forest plots and subgroup analysis. The results of the meta-analysis are shown as forest plots with heterogeneity tested by I 2 analysis. An I 2 > 50% indicated the existence of substantial heterogeneity, and subgroup analysis was further performed. All meta-analyses were conducted by Review Manager version 5.3 software (Cochrane Collaboration, Oxford, UK) provided by Cochrane. In addition, a funnel plot was generated with Stata Statistical Software version 15.0 (StataCorp LLC, College Station, TX) to evaluate the possibility of publication bias. # Results ## Selection of study A total of 502 articles were identified by searching the database and related websites. After deduplication, record screening and eligibility assessment, 28 studies remained for qualitative synthesis, as shown in. Overall, these 28 selected articles evaluated 86,835 individuals with COVID-19 infection (Supplementary. Out of the 28 studies, six studies reported irrelevant outcomes to our study aims. The last column ofdetailed the outcomes of each study. Finally, 22 studies were eligible for quantitative synthesis. ## Characteristics of selected studies All 28 articles included for qualitative synthesis were observational studies. The participants were individuals who were confirmed to have contracted COVID-19, were hospitalized for management and underwent a follow-up period of at least 28 d after study entry. Among these selected studies, 11 were conducted in the United States (USA), 9 in Asiaand 8 in Europe. The studies' sample sizes varied from 50 to 37,212. The proportion of statin uses ranged from 2% to 75%. Detailed characteristics of the included studies are presented in. All the studies were of high (n ¼ 26)or moderate quality (n¼ 2)by the NoS assessment. The most biased items were the comparability of groups and ascertainment of nonresponse rate (Supplementary. The funnel plot showed an asymmetrical distribution of the points, suggesting a lack of publication bias (p value for Egger's test ¼ 0.9933; p value for Begg's test ¼ 0.4767) (Supplementary. ## Statins and severe outcomes of covid-19 Four articles, two presenting unadjusted outcomesand another two presenting adjusted outcomes, reported the association between statins and ICU care. The meta-analysis showed that the use of statins was not significantly associated with the need for ICU care (OR ¼ 0.91, 95% confidence interval [CI]: 0.55-1.51, p¼.73, I 2 ¼ 66%). Four studies were included to evaluate how statin use affects the need for IMV in COVID-19-confirmed individuals. Three of these four studies presented adjusted outcomes regarding IMV use. The use of statins was significantly associated with a decreased risk of the need for IMV support (OR ¼ 0.81, 95% CI: 0.69-0.95, p¼.010, I 2 ¼0%). ## Statins and covid-19 mortality Eighteen studies described the mortality outcomes. Among the 18 studies, four were excluded due to incomplete data reporting, low qualityand a focus on short-term mortality (<7 d) only. A total of 14 articles were included in the final meta-analysis. Seven of these 14 articles used either propensity-matched scores or logistic regression methods to present adjusted outcomes, and another seven studies provided unadjusted data. The pooled analysis results revealed that the use of statins was linked to a decrease in the mortality rate of COVID-19 (OR ¼ 0.71, 95% CI: 0.55-0.92, p¼ .01, I 2 ¼72%). ## Subgroup analysis of statins and covid-19 mortality Since substantial heterogeneity existed in the metaanalysis of statins and COVID-19 mortality (I 2 ¼72%), we performed subgroup analysis to explore the source of heterogeneity. We identified five subgroups of studies in which statin users had even lower odds of death, including studies that (1) The heterogeneity in these five subgroups of studies was lower than that before the subgroup analyses. On the other hand, one single study that included only individuals with DM revealed that statin use was associated with increased COVID-19 mortality (OR ¼ 1.55, 95% CI: 1.07-2.25, p¼.02).describes the detailed results of the subgroup analyses. # Discussion Our study demonstrated that the use of statins was associated with a reduced mortality risk of COVID-19. When excluding one study enrolling only individuals with DM, the association between statin use and COVID-19 mortality was significant. In addition, the subgroup analysis showed that statin use prevented more deaths in studies with a CFR below 20% than in those with a CFR above 20%. This result implies that the benefit of statins seems to be greater when the COVID-19 mortality rate in the community is reduced. Our meta-analyses also revealed that statin use was linked to a reduced need for IMV but could not prevent individuals from needing ICU admission. Our finding that statins were associated with reduced COVID-19 mortality confirmed the results from previous reports. Compared with nonstatin users, statin users are usually older and have more comorbidities, which both lead to an increased risk of COVID-19 mortality. Thus, when the meta-analysis pooled the adjusted and unadjusted results, the association between statin use and mortality might not be obvious because the protective effect of statins might be countered by increased risks from host factors. In our study, statins showed benefits even when the meta-analysis pooled both the adjusted and unadjusted results, making the results more robust and convincing. One large meta-analysis performed a comprehensive systematic review and concluded statins were not associated with reduced mortality of COVID-19. However, the I 2 of the forest plot demonstrating the association of statin with mortality was as high as 90%, indicating the substantial heterogeneity across the included articles. Therefore, one essential goal of our study was to find out the subgroups that had lower heterogeneity. After the subgroup analysis, this study identified five subgroups in which statins were associated with an even lower COVID-19 mortality. The five subgroups also had a lower heterogeneity than overall participants. Our results may be helpful for participant selection in future RCTs.The measure of mortality outcome is provided for each subgroup, and the interaction p value and I 2 are provided for subgroup differences. All analyses were conducted using inverse variance and a random-effects model. One of our subgroup analyses showed that statins had survival benefits in studies with an overall CFR of less than 20% but not in those with a CFR above 20%. There are three possible explanations. First, the beneficial effect of statins was mild. When an individual bears a high risk of COVID-19 mortality, the statins' impact might be overwhelmed by the host factor and turn out to be statistically nonsignificant. Second, the effect of statins was influenced by competing medical issues. A high overall mortality rate often reflects a more severe epidemic in the community because only individuals with serious conditions can be admitted for treatment. Under such circumstances, medical resources may be insufficient. The health care system may sort medical help to those who had a better prognosis than to those who had the most severe illness. Statin users, often with more comorbidities, may have poorer outcomes than those with sufficient medical resources. Third, all confirmed COVID-19 individuals' average mortality rate in the USA was 1.82% by 6 March 2021. The higher the overall mortality rate is in a study, the lower the generalizability of the result is. As more people acquire immunity against SARS-CoV-2, either by vaccination or infection, and the health care system gains more experience in treating COVID-19, we can expect a lower overall mortality rate in the future. We believe that the analysis results focussing on low CFR studies may better apply to future conditions. According to the study results, statins were associated with a reduced need for mechanical ventilators but did not influence the possibility of ICU admission. Both of the above results had low heterogeneity. Two possible reasons may explain that. First, the criteria for the use of mechanical ventilators or ICU care might vary among different areas, hospitals and even physicians. Insufficient medical resources could also play a role in some cohorts. Second, statins provide lung protection and thus decrease the need for IMV. However, statins could not reduce other nonpulmonary events, such as cardiovascular complications or sepsis. The mechanism of lung protection is most likely via upregulation of ACE2 expression, leading to increased angiotensin (1-7) production. Although one of our subgroup analyses showed that in-hospital use of statins had a greater benefit, we cannot interpret that to mean that starting statins on admission to treat COVID-19 was helpful. According to clinical experiences, most in-hospital statin users were already taking statins before contracting COVID-19. Besides, the majority of articles included in our study emphasized the use of statin was prior to COVID-19 infection, and the results showed statin had a trend of protective effect from getting COVID-19. Retrospective observational studies had an intrinsic limitation in recording clear-cut timing of statin use among a large group of participants. However, our results at least confirmed that statin users can continue taking the medication during COVID-19 infection. There are several limitations to the study. First, all articles included in our meta-analyses were observational study designs. The baseline demographics of statin users vs. nonusers exhibited substantial differences. Furthermore, some studies reported only crude odds ratios, which might lead to an even more significant bias. Second, the type, dose and duration of statins were by no means identical across studies. The majority of the studies did not detail the prescriptions of statins. Thus, even though the current evidence showed that statins might be beneficial for patients with COVID-19, it was almost impossible to provide a clear suggestion about the drug name, dose and duration. Third, a substantial portion of participants in this study had comorbidities of diabetes or cardiovascular diseases, and might have taken other concomitant medication, such as metformin, ACE inhibitors and angiotensin II receptor blockers (ARB). Metformin has shown to be associated with reduced COVID-19 mortality, and ACEI/ARB also had close relationship with COVID-19. The influence of interaction between statins and co-medications on COVID-19 remains unclear and might impact the estimate of statins' effect. Future RCTs are needed to solve the limitations mentioned above. # Conclusions Our results demonstrated that the use of statins was associated with a reduced need for IMV and mortality rate among individuals with COVID-19. Subgroup analyses further identified individuals enrolled in studies with a lower mortality rate who benefitted more from statins. This study also confirmed that statins could be safely used during COVID-19 hospitalization.

Obesity and Cancer Metastasis: Molecular and Translational Perspectives Simple Summary: A major challenge in treating cancer is when the cancer spreads from its original site to other parts of the body, a process also known as metastasis. In order to survive, the cancer cells must communicate with their environment for survival. It is increasingly recognized that fat tissue supports the survival of metastatic cancer cells allowing tumors to form at distant sites. Obese patients therefore have a worse prognosis due to accelerated tumor spread. Preventing cancer cells from communicating with fat tissue could therefore lead to new treatments aimed at stopping this spread. In this review we discuss how dysfunctional fat tissue supports the metastatic process and evaluate new therapies and lifestyle interventions that aim to prevent the communication between cancer cells and fat tissue.Abstract: Obesity is a modern health problem that has reached pandemic proportions. It is an established risk factor for carcinogenesis, however, evidence for the contribution of adipose tissue to the metastatic behavior of tumors is also mounting. Over 90% of cancer mortality is attributed to metastasis and metastatic tumor cells must communicate with their microenvironment for survival. Many of the characteristics observed in obese adipose tissue strongly mirror the tumor microenvironment. Thus in the case of prostate, pancreatic and breast cancer and esophageal adenocarcinoma, which are all located in close anatomical proximity to an adipose tissue depot, the adjacent fat provides an ideal microenvironment to enhance tumor growth, progression and metastasis. Adipocytes provide adipokines, fatty acids and other soluble factors to tumor cells whilst immune cells infiltrate the tumor microenvironment. In addition, there are emerging studies on the role of the extracellular vesicles secreted from adipose tissue, and the extracellular matrix itself, as drivers of obesity-induced metastasis. In the present review, we discuss the major mechanisms responsible for the obesity-metastatic link. Furthermore, understanding these complex mechanisms will provide novel therapies to halt the tumor-adipose tissue crosstalk with the ultimate aim of inhibiting tumor progression and metastatic growth. Cancers 2020, 12, 3798 2 of 30Prior to exiting the primary tumor mass, tumor cells communicate with other microenvironments, termed the premetastatic niche, and this niche is selectively primed by secreted factors and extracellular vesicles to induce vascular leakage, extra cellular matrix (ECM) remodeling and immunosuppression [30]. Cancer cells also adjust the niche themselves by remodeling the ECM leading to stromal tumorigenesis [20]. Cancer patients release large numbers of cancer cells into the circulation daily, however animal studies of melanoma suggest that <0.1% of these cells metastasize [31]. The most widely studied route of dissemination is through the bloodstream (hematogenous), however metastatic cells may also migrate along nerves, lymphatic vessels, across coelomic cavities or along the basal side of endothelial cells and never enter the lumen[12]. Intravasation, the dissemination of cancer cells to organs through the lumen of the vasculature can be active or passive depending on the tumor type, microenvironment and vasculature [32]. The migration of metastatic cells into the circulation relies on chemokines and complement components that direct tumor cells through the vasculature and metabolic factors that result in an antioxidant effect [20]. Furthermore, the dissemination of circulating tumor cells (CTCs) is supported by a close association with immune cells such as activated platelets, macrophages and neutrophils [20]. When CTCs pass into small capillaries they become trapped leading to microvascular rupture or the cell undergoes extravasation [20]. Establishing a vascular network is required for metastatic colonization and this can occur though angiogenesis, co-opting existing vessels or inducing vasculogenic mimicry [20,33]. Furthermore, cancer cells can also exploit neuronal signaling pathways for growth and adaptation [20]. Cancer dormancy is an arrest phase that can occur after invasion into secondary sites and in some cancer survivors, dormant cells result in relapse long after the removal of the primary tumor [20,34]. The subsequent reactivation is governed by self-renewal pathways (e.g., Wnt, Hedgehog and Notch) and cells exhibit increased levels of stem cell associated genes [35]. While dormant cancer cells downregulate the expression of immune cell recognizable antigens, persistent host organ inflammation and the establishment of neutrophils in extracellular traps may transform dormant cells into aggressive metastasis [35,36]. In many of the steps from dissemination to colonization, the microenvironment plays a major role and this can be significantly altered by dysfunctional adipose tissue in people living with obesity.Mechanisms Linking Adipose Tissue to the Metastatic CascadeAdipocytes and AdipokinesAdipocytes secrete more than 600 soluble factors, known as adipokines, and the most well characterized are leptin and adiponectin[37]. Intra-abdominal cancers such as ovarian, colon and gastric, preferentially metastasize to the omentum, a peritoneal organ largely composed of adipocytes, suggesting that adipocytes significantly contribute to the metastatic cascade[38]. Omental adipocytes promote migration and invasion of ovarian cancer cells by secreting cytokines[39]. Neutralization of these cytokines reduced in vivo homing of ovarian cancer cells to mouse omentum, suggesting that adipocytes promote the early stages of metastasis[39]. In addition, ovarian cancer cells metabolically adapt to the increased availability of lipids by utilizing energy from fatty acids for growth[39]. Fatty acid binding protein 4 (FABP4) was strongly expressed at the adipocyte-cancer cell interface and pharmacological inactivation of FABP4 decreased cancer cell lipid accumulation, invasiveness and omental metastasis[39]. Circulating levels of FABP4 are markedly increased in obese individuals due to release from an expanded adipose tissue depot and FABP4 can induce mammary tumor stem cells by enhancing ALDH1 activity via IL-6/STAT3 signaling[40,41]. In addition, FABP4 promoted aggressive acute myeloid leukemia (AML) in obesity by enhancing aberrant DNA methyltransferase 1 (DNMT1)-dependent DNA methylation of tumor cells[42]. Upon interaction with cancer cells, adipocytes dedifferentiate into preadipocytes or are reprogrammed into cancer-associated adipocytes (CAAs), which resemble fibroblasts and have dispersed lipid droplets[43]. In breast cancer, matrix metalloproteinase (MMP)-11 is induced in adipocytes by adjacent invading cancer cells. In the presence of MMP-11, the activated adipocyte dedifferentiates into a preadipocyte fibroblast-like cell, # Introduction The metabolic and cardiovascular risks of obesity are well known. However, it is estimated that 40% of all cancer deaths are also attributable to obesity. Indeed, globally, excess body weight is third behind smoking and infection as an attributable risk factor for cancer, and second to smoking in Western populations. Obesity adversely effects cancer in two ways, (i) by promoting carcinogenesis resulting in a higher cancer incidence and (ii) cancer progression resulting in an increased risk of mortality. In breast cancer, for example, obesity is only associated with an increased incidence of post-menopausal breast cancer, whilst obesity is a risk factor for progression in all breast cancer subtypes. The global obesity rate in women is projected to reach 21% by 2025 and this is particularly alarming considering that 55% of all female cancers have an obesity associated mechanism. Central obesity, resulting from the overgrowth of visceral white adipose tissue (WAT), has been specifically linked to cancer progression. While diet is undoubtedly important in obesity, animal models have indicated that WAT overgrowth directly promotes cancer progression irrespective of diet. Epidemiological studies have demonstrated a compelling association between cancer risk and obesity. Analysis of the association between body mass index (BMI) and early stage breast cancer outcomes in the Danish Breast Cancer Cooperative Group (n = 53,816 women) revealed that obese women have a 46% higher risk of developing distinct metastasis at 10 year follow up compared to normal weight women. Furthermore, a meta-analysis of 82 studies found a 41% and 35% higher risk, respectively, of all-cause mortality and breast cancer specific mortality in obese women compared to normal weight women. An umbrella review of 204 meta-analyses revealed a strong association between obesity and gastrointestinal cancers including esophageal adenocarcinoma (OAC). OAC was notable for a progressive increase in risk ratio (RR) for each 5 kg/m 2 increase in BMI (RR 4.8 for a BMI ≥ 40 kg/m 2 ) suggesting a dose-response effect. The top two cancers demonstrating very strong associations with BMI are endometrial (RR 1.48) and OAC (RR 1.45). In obesity, white adipocytes become hypertrophic and hyperplasic, which results in physiologic changes, including elevated free fatty acids (FFAs) and triglycerides, increased blood glucose and insulin resistance. Obese adipose tissue increases production of proinflammatory cytokines, e.g., tumor necrosis factor (TNFα), interleukin-6 (IL-6), interleukin-1β (IL-1β) and adipokines (e.g., leptin). Metastasis is the primary cause of cancer morbidity and mortality and efforts to unravel the molecular mechanisms linking dysfunctional adipose tissue and the ability of tumor cells to acquire metastatic properties will lead to the discovery of novel targets for metastasis. Here we review the recent findings regarding the molecular processes underpinning the impact of adipose tissue on cancer metastasis and potential therapeutic interventions. ## The metastatic cascade The "hallmarks of cancer" define characteristics that are critical for cellular transformation. Among the hallmarks there is only one defining factor, invasion, which distinguishes a malignant and benign tumor. The metastatic cascade begins with local invasion before progressing to intravasation, arrest at distant organs, extravasation, micro metastasis formation and finally metastatic outgrowth. In the early stages of tumor development, the primary tumor cell mass typically has an expansive phase in the absence of invasion, encapsulated in a dense fibrous network (i.e., desmoplasia). A subset of neoplastic cells acquire the ability to escape through the basement membrane and detach from the primary tumor. The dissemination of cancer cells is a consequence of chromosomal instability that causes continuous errors in chromosome segregation during mitosis. This in turns leads to the rupture of micronuclei and the secretion of genomic DNA into the cytosol, which activates DNA sensing pathways and NF-κB signaling. In addition, epithelial-mesenchymal transition (EMT) is a transdifferentiating process, which permits epithelial cells to attain a mesenchymal phenotype with migratory potential. Spontaneous EMT in primary tumor cells can be triggered by hypoxia, metabolic stressors and matrix stiffness . Although some studies have cast doubts on the necessity of EMT during metastasis , there is evidence for cells expressing both epithelial and mesenchymal markers within the primary tumor, circulation and at a secondary metastatic site . Therefore EMT is being increasingly understood as a spectrum of transitional stages between epithelial and mesenchymal phenotypes rather than a binary choice between an epithelial or mesenchymal phenotype . There is evidence that cells in different EMT stages prefer certain microenvironments, for example, metastatic cells with a predominating mesenchymal phenotype proliferate near endothelial and inflammatory cells . These tumor cells release factors to attract immune cells and stimulate angiogenesis, thus promoting an inflammatory and highly vascularized niche . Although EMT is required for metastatic dissemination, the opposite process of mesenchymal to epithelial transition (MET) is required for metastatic colonization of distant sites . There is also substantial evidence that disseminated tumor cells express stem cell markers, such as aldehyde dehydrogenase (ALDH), and functionally these cells, highly enriched in stem cells markers, have an enhanced ability to cause metastasis . Furthermore, genome wide analysis of both cells undergoing EMT and circulating tumor cells has revealed a similar transcriptome to primary cancer stem cells (CSCs) thus indicating an overlapping subpopulation . In pancreatic cancer, primary CD133 + CSCs demonstrated classic CSC characteristics such as tumor initiation and chemoresistance . However, at the invasive front of the tumor CD133 + cells are enriched for CXC-chemokine receptor 4 (CXCR4) and the CD133 + CXCR4 + population is more migratory than CD133 + CXCR4 - . Moreover, patients with increased CD133 + CXCR4 + cells had more metastatic disease . This indicates that microenvironmental cues within the tumor can trigger heterogeneity in CSCs and CD133 + CXCR4 + and CD133 + CXCR4are not a distinct subpopulations but a gradient of stemness phenotypes . CSCs are more resistant to chemotherapy due to higher expression of multidrug resistance (MDR) or detoxification proteins such as aldehyde dehydrogenase (ALDH) [29]. which can sustain cancer cell invasion. Bone marrow adipocytes constitute approximately 15% of bone marrow volume in young adults, rising to 60% by the age of 65 years old. They have a distinctive phenotype, which resembles both white and brown adipose tissue, and they secrete fatty acids, cytokines and adipokines, which influence the whole bone microenvironment. The bone provides a supportive microenvironment for both solid tumor and hematological metastasis, including breast, prostate and multiple myeloma and bone metastatic cancers primarily occur in older adults whose bone marrow is heavily populated by adipocytes. Similarly to the omentum, cancer cells are attracted to the adipocytes in the metabolically active red marrow and this creates a niche in the bone marrow for disseminated cancer cells to establish and progress. In addition, leukemic stem cells expressing the fatty acid transporter CD36, induce lipolysis in gonadal adipose tissue to support their metabolism and evade chemotherapy. Lipids can also be trafficked between bone marrow adipocytes and cancer cells by upregulation of FABP4 and fuel growth and invasiveness in metastatic tumor cells. In addition, bone marrow adipocytes have been shown to promote the Warburg phenotype in metastatic prostate cancer cells through oxygen independent HIF-1α activation. Bone marrow adipocytes are a major source of circulating adiponectin, much greater than WAT. Adiponectin is reported to suppress many elements of the early metastatic cascade including adhesion, invasion, migration and stem cell properties via numerous signaling pathways including WNT, NF-κB and JAK/STAT. In advanced cancer associated with cachexia, hyperadiponectinemia has been observed. In addition, increased adiponectin signaling in dendritic cells can blunt anti-tumor immune responses in patients with metastatic disease. However, the late increase in adiponectin has very little influence on the course of the disease, as its role is thought to be more prominent in early metastatic spread. During the development of obesity, preadipocytes differentiate incorrectly leading to hypoxia and the induction of hypoxia induced factor-1 (HIF-1). This inhibits the expression of adiponectin and increases the expression of leptin, resulting in a reduced adiponectin to leptin ratio in obesity-associated adipose tissue. A high leptin to adiponectin ratio has been reported to increase the risk of postmenopausal and triple negative breast cancer (TNBC) progression. Leptin is another adipokine important in tumor progression and secretion of leptin is increased in CAAs compared to mature adipocytes. Leptin levels are increased in the plasma of post-menopausal breast cancer patients, which correlated with a higher grade, advanced tumor stages and presence of distant metastasis. Leptin exerts it effect through the transmembrane leptin receptor and only the full length isoform, ObRb, contains the intracellular domain required for JAK/STAT signaling. ObRb was significantly overexpressed in metastatic lymph nodes compared to the primary tumor in ER-breast cancer patients. Furthermore, TNBC patient derived xenografts (PDX) grown in the presence of primary adipose stem cells (ASCs) isolated from obese donors (obASCs) had increased HLA1 + human tumor cells and CD44 + CD24 − CSCs in the peripheral blood and metastasis compared to ASCs from lean women. In addition, the knockdown of leptin expression in obASCs suppressed the prometastatic effect. There is a plethora of evidence that leptin induces cell migration and invasion in breast cancer via JAK/STAT3 signaling. In addition, the Notch signaling pathway is a key regulator of leptin induced cell migration in breast cancer obesity models. Leptin has also been shown to promote the CSC phenotype though STAT3 activation and this in turn recruited a histone methyl transferase causing a repression of miR-200c by epigenetic silencing and the expansion of CSCs. Autotaxin (ATX) is an adipocyte-derived lysophospholipase D that catalyzes the hydrolysis of circulating or cell associated lysophosphatidylcholine (LPC) to the bioactive lipid lysophosphatidic acid (LPA). Some tumor cells, such as in melanoma, glioblastoma and thyroid cancer, directly secrete ATX leading to a chronic inflammatory state and decreased acquired immune response. In contrast, other tumor cells, such as breast, ovarian and pancreatic do not produce ATX and instead the tumor microenvironment is the primary source. Interestingly, the ATX gene (ENPP2) was the second most upregulated gene in breast CSCs treated with paclitaxel whilst the LPP2 gene (PLPP2) was downregulated, indicating CSCs may favor an LPA-enriched microenvironment. In a tissue microarray (TMA) of metastatic breast cancer, stromal ATX was highly expressed in bone metastasis. In mice approximately 40% of ATX is produced by adipocytes and this increases when mice are fed a high fat diet. Similarly, in humans ATX production increases in obesity, particularly in inflamed adipose tissue, and contributes to comorbidities including insulin resistance, hepatic steatosis and atherosclerosis. In breast cancer cells both ATX and LPA are associated with mobility and invasive capacity via the JAK/STAT3 pathway or PI3K/MAPK pathways. Therefore cross talk between adipose tissue derived ATX and tumors cells is a potential mechanism for tumor progression. Indeed preclinical studies of an ATX inhibitor, ONO-843050 suppresses tumor growth and 60% of lung metastasis in a breast cancer mouse model. Targeting other molecules in the ATX-LPA signaling pathways also results in decreased breast cancer metastasis formation in murine models. The physiological upregulation of ATX occurs in response to inflammation and chronic activation of ATX-LPA signaling occurs in diseases such as pulmonary fibrosis, rheumatoid arthritis and inflammatory bowel diseases. A first in class ATX inhibitor, GLPG1690, attenuated idiopathic pulmonary fibrosis in a Phase IIa clinical trial (NCT02738801) and two Phase III clinical trials are currently underway for this indication (NCT03711162; NCT03733444). In a preclinical breast cancer model, GLPG1690 acted synergistically with chemotherapy and radiotherapy to improve outcomes. Considering the role of ATX in regulating CSCs and mobility, targeting ATX with inhibitors such as GLPG1690 may also prove to be beneficial in targeting metastasis, particularly in patients living with obesity, by preventing adipose tissue cross talk. ## Immune cells and inflammatory factors CAAs secrete more inflammatory factors, such as monocyte chemoattractant protein (MCP-1), RANTES, IL-1β, IL-6 and TNFα than "normal" adipocytes and this can promote invasion and metastasis formation. Furthermore, the recruitment and activation of immune cell subsets, particularly M1 macrophages, in obese WAT increases local and systemic levels of proinflammatory cytokines. MCP-1 is elevated in obesity and secreted by many cells including tumor cells, fibroblasts, tumor infiltrating monocytes and endothelial cells. In cancer cells, MCP-1 induces the expression of NOTCH1 and subsequently promotes the activity of CSCs and neovascularization. RANTES expression in the peritumoral adipose tissue of women with TNBC correlated with lymph node and distant metastasis. Furthermore, the RANTES inhibitors, maraviroc and vicriciroc, reduced invasion and pulmonary metastasis in a preclinical tumor model of breast cancer. IL-6 is a pleotropic cytokine involved in immune regulation and tumorigenesis. One third of total circulating IL-6 originates from adipocytes and circulating levels are correlated with adiposity. When adipocytes are cocultured with breast cancer cells, adipocytes increase secretion of IL-6, which in turn promotes invasion and migration of tumor cells. Furthermore, IL-6 also plays a critical role in the biology of CSCs through activation of Notch and JAK/STAT signaling. In addition to classical IL-6 signaling, IL-6 trans signaling is a major driver of obesity associated hepatocellular carcinoma (HCC), through inhibition of p53 induced apoptosis and enhanced angiogenesis. IL-6 also promoted HCC progression via upregulation of osteopontin (OPN), a secretory ECM protein involved in the maintenance of the stemness phenotype. The inflammasome is a highly regulated protein complex that triggers caspase-1 activation and subsequent secretion of IL-1β and IL-18. Obesity related factors, such as cholesterol crystals and fatty acids (palmitate and ceramide), can lead to unchecked activation of the inflammasome. IL-1β expression in primary tumors is a potential biomarker for predicting breast cancer patients who are at increased risk of developing bone metastasis. Furthermore, in vitro studies indicate that tumor cells expressing high levels of IL-1β specifically home to and colonize the bone. A persistent increase of circulating levels of TNFα occurs in obesity, mainly due to the elevated number of M1 macrophages in obese WAT. TNFα stimulates the secretion of MMPs in epithelial tumors and enhances EMT to promote invasion and migration of tumor cells. Furthermore, TNFα increases liver metastasis through inducing expression of cell adhesion molecules, such as ICAM-1, E-selectin and VCAM-1, on liver specific endothelial cells, and thus enhancing tumor cell arrest and transendothelial migration. Crown like structures (CLSs) are a histological feature of dead or dying adipocytes surrounded by macrophages and they are increased in obesity associated adipose tissue. CLS are associated with free fatty acid (FFA) release, NF-κB activation and the generation of a proinflammatory microenvironment. They are best characterized for their role in the initiation and progression of breast cancerbut they are also reported to play a role in endometrial cancer, prostate cancerand non-alcoholic steatohepatitis (NASH)-a major risk factor for HCC. In breast adipose tissue CLS are not only associated with inflammation but they also drive aromatase activity resulting in an increased ratio of estrogen:androgen in blood and local tissues. The expression of programmed death-ligand 1 (PD-L1) in adipocytes prevents the antitumor function of cytotoxic CD8 + T cells. It is therefore not surprising that treatment with anti PD-1/PD-L1 immunotherapy in patients with a BMI > 25 (i.e., overweight/obese) have improved clinical outcomes compared to normal weight patients. CSCs will only proliferate in specific tumor environments indicating that environmental stimuli are critical to preserve their phenotypic plasticity, to protect them from the immune system and to facilitate metastatic potential. High levels of proinflammatory cytokines from obese adipose tissue can stimulate CSC properties. TNFα enhances the CSC phenotype in numerous cell types and is associated with upregulation of stem cell related genes, chemo resistance and tumorigenesis. Furthermore, TNFα upregulates TAZ (a Hippo pathway effector) and Slug (an EMT mediator), which increase breast CSCs through both canonical and non-canonical NF-κB signaling. Analysis of TNBC patient datasets reveals high tumor expression of the epigenetic reader methyl-CpG-binding domain protein 2 (MBD2), specifically the alternative splicing variant 2 (MBD2_v2) expression and high relapse rate and high BMI. It is postulated that obesity drives high reactive oxygen species (ROS) levels, which subsequently promotes MBD2_v2 expression and an expansion of the CSC fraction. ## Angiogenesis The processes of angiogenesis and adipogenesis are closely linked. When preadipocytes differentiate into adipocytes and become adipose tissue, new blood vessels are also formed. Conversely inhibition of adipocyte differentiation also reduces angiogenesis. In breast cancer, leptin upregulates all components of the IL-1 system (IL-1α, IL-1β, IL-1Ra and IL-1R tI) and both leptin and IL-1 together promote angiogenesis through expression of VEGF/VEGFR. Furthermore, leptin upregulation of VEGF/VEGFR2 was impaired by IL-1 signal blockage. In addition, obesity leads to an increase in tumor infiltrating macrophages with activated NLRC4 inflammasome and increased IL-1β production in breast tumors. This leads to a NLRC4/IL-1β dependent upregulation of adipocyte derived angiopoietin-like 4 and enhanced obesity associated tumor angiogenesis. Anti VEGF therapy has fallen short of expectations, particularly in breast cancer where the FDA revoked approval for bevacizumab because of a lack of overall survival benefit. Anti VEGF therapy resistance is partly driven through expression of proinflammatory and other alternative angiogenic factors, many of which are also increased in obesity. Furthermore, breast cancer patients with obesity are less sensitive to anti VEGF treatment and they have increased systemic concentrations of IL-6 and fibroblast growth factor-2 (FGF-2). The elevated IL-6 was associated with increased IL-6 production from adipocytes and myeloid cells within tumors and IL-6 blockage abrogated obesity induced resistance to anti VEGF therapy at both primary and metastatic sites. Vasculogenic mimicry (VM) is a tumor vascular system that is independent of angiogenesis of endothelial cells, and is associated with both poor survival in multiple tumor types and anti VEGF therapy resistance. Notably, the adipose tissue secretome has been shown to induce melanoma cells to arrange in 3D vessel like structures, characteristic of vasculogenic mimicry, supporting a role of adipose tissue in this process. ## Metabolic repogramming An essential function of adipocytes is energy mobilization and therefore a metabolic interaction between cancer cells and adipocytes is not surprising. The Warburg effect suggests that due to mitochondrial dysfunction, malignant cells prefer to produce adenosine triphosphate (ATP) via glycolysis instead of oxidative phosphorylation, even in the presence of oxygen. In parallel, cancer cells are able to use alternative sources of energy such as amino acids and lactate from the microenvironment. Bone marrow adipocytes promoted the Warburg phenotype by increased expression of glycolytic enzymes, increased lactate production and decreased mitochondrial oxidative phosphorylation in metastatic prostate cells by paracrine signaling. The "reverse Warburg effect" theory proposes that cancer cells induce oxidative stress in the neighboring stromal cells by secreting ROS and triggering aerobic glycolysis and production of high energy metabolites, especially lactate and pyruvate. These metabolites are then transported through the "lactate shuttle" to sustain the anabolic needs of adjacent cancer cells. The effect has been mainly described in stromal fibroblast cells, however given that ASCs and CAA are fibroblast like cells, it is likely they are also important contributors. Indeed it has been reported that the "reverse Warburg effect" was induced during the co-culture of adrenocortical carcinoma cells with ASCs. Ketone bodies are another catabolite produced and released by glycolytic adipocytes and they are an ideal substrate for ATP production by driving oxidative mitochondrial metabolism leading to enhanced tumor invasiveness. A key characteristic of CAA is their loss of lipid content. The FFAs released by adipocytes after lipolysis are stored in tumor cells as triglycerides in lipid droplets. Tumor cells then release FFAs from lipid droplets though an adipose triglyceride lipase dependent lipolysis (ATGL) pathway. ATGL is upregulated in tumors on contact with adipocytes and it correlates with aggressiveness by stimulating tumor cell invasion. The FFAs also act as structural units for newly synthesized membrane phospholipids and cancer cell membranes become enriched with saturated and/or mono unsaturated fats leading to changes in membrane dynamics. This results in cancer cells that are more resistant to oxidative induced cell death and reduced the uptake of drugs. Advanced metastatic melanomas frequently grow in subcutaneous tissues largely composed of adipocytes. Adipocyte derived lipids are transferred to melanoma through the lipid transporter FATP1 and a small molecule inhibitor of FATPs reduced melanoma growth and invasion. Furthermore in AML, leukemic blasts activate lipolysis in neighboring bone marrow adipocytes leading to the transfer of lipids to the blast through FABP4. Amino acids such as glutamine, glycerine, serine and proline also have important roles in the asymmetric metabolism of amino acids between cancer and stromal cells. Glutamine is a pivotal source of the TCA cycle intermediates and ATP in cancer cells, and the substrate of the antioxidant glutathione. Stromal cells within the tumor microenvironment harness carbon and nitrogen from non-canonical sources to synthesize glutamine and it is used by the tumor cells to promote growth and metastasis. Glutamine is downregulated in obesity and is inversely associated with proinflammatory gene expression and macrophage infiltration. Pancreatic ductal adenocarcinoma (PDAC) cells rely on glutamine utilization to fulfill their metabolic requirements and it is the most depleted amino acid within the PDAC microenvironment. Glutamine deficiency leads to the induction of EMT through the upregulation of the master EMT regulator Slug. In addition, as glutamine levels decline, tumor cells become more reliant on asparagine for proliferation and protein synthesis. Asparagine affects the metastatic cascade at multiple stages. At the primary tumor level, asparagine promotes EMT and intravasation. In the circulation asparagine helps circulating tumor cells survive shear and oxidative stress whilst at a distinct metastatic sites, asparagine facilitates cell growth and survival by inducing glutamine synthetase (GLUL) expression and glutamine biosynthesis. Citrulline and nitric oxide are generated by tumor cells by catabolizing the semi essential amino acid arginine. Nitric oxide facilitates glycolytic activity and suppresses oxidative phosphorylation to promote proliferation. Citrulline is secreted into the ECM and is taken up by stromal adipocytes, before being converted back into arginine and released for cancer cells. Depriving tumor cells of arginine has cytotoxic effects through apoptosis or autophagy depending upon the tumor type, and decreasing the ability of tumor cells to migrate and adhere to the ECM. Arginine dependent migration requires arginine to be metabolized by two major enzymes, arginase (ARG1) and nitric oxide synthase (NOS). In HCC, higher expression of ARG1 is positively correlated to aggressive tumor growth and poor disease free survival. In vitro studies revealed that overexpression of ARG1 enhanced arginase activity leading to multiple processes that contribute to progression including increased cell viability, migration, invasion and EMT. Obesity coupled with PDAC results in accelerated tumor growth and enrichment in pathways regulating nitrogen metabolism. The mitochondrial form of arginase (ARG2) that hydrolyzes arginine into ornithine and urea is induced upon obesity and is accompanied by PDAC growth and increased nitrogen flux from 15N-glutamaine into the urea cycle, the principle pathway for ammonia detoxification. Infusion of 15N-arginine in murine models demonstrates a shunting of arginine catabolism away from the urea cycle into creatine synthesis, resulting in ammonia accumulation specifically in obese tumors. The biological consequences of ammonia accumulation in the tumor microenvironment is not fully understood but it has been shown to directly generate amino acids through glutamate dehydrogenase activity. Hyperinsulinemia is a hallmark of chronic obesity and insulin stimulates the hepatic synthesis of the peptide IGF-1. Three quarters of breast cancer patients show activation of insulin/IGF-1 signaling and this rises to 87% in patients with invasive breast cancer. In preclinical models, blocking IGF in combination with paclitaxel significantly reduced tumor cell proliferation and lung metastasis. Due to the frequent dysregulation of the IGF system in cancer, various components of this system became attractive anticancer targets. However, clinical trials using IGF-1 receptor blocking antibodies failed to meet expectations, except in a small number of malignancies. More recent developments reveal that dysregulation of the IGF system results in a substantial expansion of the cancer stem like subpopulation by supporting EMT and self-renewal signaling pathways. IGF signaling regulates these pathways in multiple ways though i) stimulation of the transcription factors of the ZEB and the Snail family implicated in the EMT program, ii) interacting with pluripotency transcription factors (e.g., Oct-4, SOX2, Nanog, p53 and HMGA1 proteins) and iii) regulation of development signaling factors (e.g., Wnt/β-catenin, Notch and Shh pathways) classically involved in cell stemness. Intriguingly, in HER2+ breast cancer patients, high IGF-1 in normal weight patients showed a superior recurrence free survival compared to low IGF-1. In contrast, high IGF-1 in overweight patients was associated with a reduced recurrence free survival. Obese mice have a heightened inflammatory response in the liver and an increased incidence of metastatic colon carcinoma cells to the liver. Moreover, liver inflammation induced by obesity was abrogated in liver specific IGF-I deficient mice leading to a significant reduction of in liver metastasis. Furthermore, IGF-1 promotes neutrophil polarization to a tumor promoting phenotype and the induction of a prometastatic microenvironment in the liver. ## Extracellular matrix In addition to adipocyte hypertrophy and dysregulated lipid metabolism, heightened inflammation, hypoxia and abnormal angiogenesis, obesity is also associated with ECM remodeling. Once a primary tumor is established, cells migrate and invade to form satellite tumors within centimeters of the original tumor mass and/or disseminate through the lymph nodes and vasculature to form secondary macrometastases at distance sites. This progression sequence is dependent on the ability of cancer cells to traverse the ECM. The adipose tissue ECM is a three-dimensional, non-cellular structural support of the numerous cell types that reside in the adipose tissue. It is composed primarily of collagens, fibronectin and to a lesser extent lamins that are supplied by a number of resident cell types including fibroblasts, adipocytes and preadipocytes. In obesity, the highly dynamic adipose tissue ECM is constantly undergoing remodeling and reorganization to accommodate increased adipocyte numbers and adipocyte hypertrophy. A rapid increase in adipose tissue volume can result in regional hypoxia, which triggers excess deposition of fibrillar collagens by adipocytes and myofibroblasts, immune cell infiltration, adipose tissue fibrosis, a desmoplastic stroma and increased tissue stiffness, with overall behavior described as similar to "a wound that never heals". This state of chronic low-grade inflammation within the adipose tissue drives obesity-associated pathologies including diabetes, cardiovascular diseaseand cancer. Indeed, it is very similar to the microenvironment of a thriving tumor mass and thus trophic cancer cells that home to the adipose tissue are well supported by these suitable surroundings. Fibrosis is a hallmark of cancer, and desmoplasia within the tumor microenvironment, is a marker of poor prognosis in cancerand can negatively impact drug delivery. In the case of PDAC, obesity is associated with aggressive tumors with poor prognosis, and adipocyte accumulation in the malignant pancreas. Obesity-induced accumulation of high adipocyte numbers in the pancreas has been shown to induce inflammation and excessive accumulation of ECM components, i.e., desmoplasia, which promotes tumor progression and resistance to chemotherapy. Adipose tissue is the main component of the breast cancer microenvironment, crosstalk between the breast cancer cells and adipocytes or other adipose stromal cells stimulates the secretion of even larger quantities of ECM proteins, increasing matrix stiffness and scar formation, further enhancing EMT and local invasion of tumor cells. Within the adipose tissue, invading breast cancer cells manipulate adipocytes to form fibroblastic CAAs that secrete large volumes of ECM proteins including collagen I, III and IV, and the cleavage product of collagen IV, endotrophin, which is associated with breast cancer metastatic spread. Furthermore adipocyte collagen IV has been shown to play a role in the early stages of tumor growth in breast cancer. Recently, it has been shown that breast cancer secreted PAI-1 can stimulate CAA collagen biogenesis and reorganization via the induction of a lysyl hydroxylase protein, PLOD2, facilitating the migration of breast cancer cells along aligned collagen fibers, in vitro and in vivo, further promoting metastasis. Additionally, tumors growing in the adipose tissue-rich microenvironment can induce morphological and functional changes in ADSCs so that they differentiate into a CAF-like myofibroblastic phenotype. Breast cancer cells can induce the differentiation of ADSCs to CAFs involving a mechanism dependent on TGF-β, and these ASC-derived CAF-like cells can promote breast cancer cell motility and invasion in vitro, and expressing high levels of stromal-derived factor 1 (SDF-1), a chemokine associated with a more aggressive and invasive cancer phenotype. During obesity-induced fibrosis, as adipocytes become encased in the rigid ECM, necrosis ensues and dysfunctional adipocytes stimulate the recruitment of macrophages to the site, histologically forming CLS around the dying adipocytes. The contribution of these activated macrophages to ECM production most likely occurs through their effects on other cell types. They are a major source of TGF-β, PDGF and other chemokines that attract and activate more ECM protein producing fibroblast type cells to the adipose tissue. Finally, fibrosis dynamics are tightly regulated by the metalloproteinase (MMP) protein family, which cleave collagenous fibers, enabling matrix remodeling. There are many MMPs associated with obesity, in particular MMP-11 (also known as stromelysin-3/ST-3) has been shown to be overexpressed by adipocytes as a result of stimulation by invading breast cancer cells. MMP-11 is important for collagen VI folding and it is also known to negatively regulate adipogenesis and can dedifferentiate adipocytes so that they can acquire a more fibroblast-like phenotype that benefits the invading tumor cells via involvement in adipose tissue fibrosis and ECM remodeling. ## Extracellular vesicles In addition to the milieu of adipose tissue secreted factors discussed in the previous sections, there is now evidence highlighting the role that adipose tissue derived extracellular vesicles (EVs) play in guiding and enhancing the metastatic process. EVs are lipid membrane enclosed particles, measuring on the nanometer scale, that can be classified as either minute exosomes (<100 nM) or larger microvesicles (<1000 nM), which facilitate the horizontal transfer of cellular cargo, including nucleic acid, proteins, lipids and metabolites between communicating cells. It is not clear whether obesity alters the content of EVs produced by adipocytes, however in the obese setting larger quantities of adipocyte-derived EVs are secreted compared to lean conditions. While the role of cancer cell-derived EVs in manipulating cells in the tumor microenvironment including adipocytes is well established, very little attention has been given to the bidirectional role EVs play in adipocyte-cancer cell communication, and particularly the influence of adipocyte-derived EVs on tumor cell behavior. Nevertheless, a small number of recent studies demonstrate a link between adipocyte EVs and tumor progression in obesity-driven lung cancer, breast cancerand melanoma. One way in which adipocytes can promote tumor progression is through metabolic cooperation, by providing a local supply of fatty acids for the process of fatty acid oxidation (FAO) within tumor cells, an emerging favorable metabolic pathway that enhances tumor invasiveness, proliferation and stem cell properties. In the later stages of tumor progression once the tumor cells have invaded the adipose tissue, secretion of tumor-derived soluble factors can stimulate adipocyte lipolysis and extracellular release of FFAs into the surrounding microenvironment. Aside from direct release of FAs from adipocytes, EVs released by adipocytes can also be used as a method to transfer molecules including FA substrates and the protein machinery required for FAO to cancer cells either locally or over larger distances, e.g., through the circulation and other tissues. Epidemiological studies have identified a link between melanoma aggressiveness and obesity. Aptly, subcutaneous adipocytes are one of the main components of the tumor microenvironment of invading melanomas and indeed melanoma cells have been demonstrated to internalize naïve adipocyte (i.e., not previously exposed to cancer cells)-derived EVs, resulting in amplified FAO and an enhanced migratory and invasive tumor cell phenotype. Lazar et al. demonstrated that melanoma cells cultured with adipocyte secreted EVs had an increased ability to form lung metastases in mice xenograft models, with a concomitant upregulation of tumor cell FAO. As previously mentioned obese-derived adipocytes secrete higher numbers of EVs compared to their lean counterparts, thus when comparing the effect of adipocyte EVs derived from obese versus lean conditions, equal concentrations of EV preparations were applied to melanoma cells. Noticeably, in obese conditions only adipocyte-derived EVs elicit a heightened effect on melanoma migration, in addition to enhancing clonogenicity and metastatic potential. Thus differences in the cargo content of these EVs as opposed to the sheer number of vesicles are most likely responsible for this heightened effect. Recent in-depth labeling experiments indicate that around 30% of the proteins within the adipocyte EVs are sufficiently transferred to melanoma tumor cells, and these include proteins involved in EV transport, the transport and storage of FAs, mitochondrial FAO and oxidative phosphorylation. Efforts to understand how an obese state heightens the effect of adipocyte EVs on FAO-induced functions of melanoma cells have revealed that the level of FAO enzymes in melanoma tumor cells is unaltered following uptake of obese versus lean derived EVs. In contrast adipocyte EV supply of FAs and subsequent trafficking to melanoma cells was increased under obese conditions, resulting in enhanced substrate availability and FAO, and the altered mitochondrial dynamics that is critical to melanoma cell migration and invasion. Thus in the obese setting, it is the increased transfer of substrate (e.g., FA) and not machinery (FAO-related enzymes) that enhance FAO in recipient melanoma cancer cells. In whole adipose tissue, aside from mature adipocytes, EVs are found in the supernatant ASCs. While adipocyte EVs are linked to enhanced tumor invasiveness and metastatic potential via lipid metabolism, current literature suggest ADSC secreted EVs play a role in angiogenesis, immune modulationand tumor development. When exosomes secreted from the preadipocyte cell line, 3T3-L1, are injected into the mammary fat together with breast cancer cells, primary tumor initiation and growth is enhanced. Of particular interest in this review, ASC derived exosomes have been shown to promote proliferation and migration, at least in part through the modulation of Wnt/β-catenin signaling, a key pathway in tumor stemness, EMT and metastasis. ASCs are abundant in the microenvironment of highly metastatic osteosarcoma. Recently ASC exosomes has been shown to increase proliferation, invasion and migration of osteosarcoma cells in vitro, and growth and metastasis in vivo, via an induction of COLGALT2, a prometastatic gene that subsequently activates downstream EMT targets vimentin and MMP-2 and -9. Studies regarding adipose tissue secreted exosomes, be it adipocytes or ASCs, are limited to a small number of studies and a small number of tumor types. Further research is required to understand the role, if any, of adipose tissue-derived EVs in other cancer types, which share an intimate relationship with adipose tissue and are driven by obesity. ## Targeting metastasis through adipose tissue-tumor interactions Obesity-associated cancer is a substantial public health problem worldwide and correcting the disharmony within dysfunctional adipose tissue represents an opportunity to halt disease progression, as summarized in. Due to the importance of adiponectin in the progression of several cancer types, adiponectin receptors agonists have been developed. An adiponectin-based short peptide named ADP 355 has high affinity with AdipoR1 and regulates canonical adiponectin signaling pathways (i.e., AMPK, Akt, STAT3, ERK1/2) to halt tumor growth. Adiponectin has a larger role in early stage metastatic processes and there are concerns that its agonists may blunt antitumor immunity. Therefore cancer stage is an important consideration for adiponectin-based agonist therapy. Interestingly, adiponectin may also be modulated with dietary and lifestyle factors. Daily fish intake, omega 3 and fiber supplementation have been shown to increase blood levels of adiponectin. Furthermore, moderate intensity aerobic exercise was shown to elevate adiponectin levels to 260% within one week. Inhibitors of leptin, another important adipokine, are also in development. Conditioned media (CM) from breast adipocytes significantly increased mammosphere formation, a marker of breast CSCs, and depletion of leptin from the CM completely abrogated this effect. In addition, blocking leptin signaling using a full-length leptin receptor (ObR) antagonist also reduced mammosphere formation indicating a potential therapeutic target to block stromal-tumor interactions driving breast CSC-mediated disease progression. The ObR antagonist, Allo-aca, a short leptin-based peptidomimetic inhibits leptin induced tumor cell proliferation and angiogenesis. In addition, it has good biodistribution to the brain and could potentially target brain metastasis. Adipose tissue significantly contributes to inflammatory cytokines in the tumor microenvironment that may be targeted therapeutically to treat metastasis. Agents targeting IL-6 or the IL-6 receptor have already received FDA approval for the treatment of inflammatory conditions or myeloproliferative neoplasms and are being evaluated in solid tumors. However, despite promising preclinical activity, targeting IL-6 alone in unselected cancer patient populations has not shown benefit to patient outcomes. Therefore, more selective formulations and selecting potentially sensitive patient populations, such as obese patients, are required. A novel-CD44 antibody mediated liposomal nanoparticle loaded with anti-IL6R antibody demonstrated specific antimetastatic efficacy in a preclinical mouse model by inhibiting IL-6/STAT3 signaling in CSCs and also reducing the SOX2 + and CD206 + cells in the tumor microenvironment of lung metastasis, thus demonstrating dual inhibitory activity on CSCs themselves and the metastatic niche microenvironment. Hyperinsulinemia in obesity induces insulin growth factor (IGF)-1 signaling, which promotes epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) though ZEB and SNAIL transcription factors leading to an increase in pluripotency transcription factors (OCT4, SOX2 and NANOG) and the developmental signaling pathways (Wnt and NOTCH). Interleukin 1β (IL-1β) promotes promote tumor cell homing to the bone marrow and angiogenesis through vascular endothelial growth factor (VEGF) signaling. Interleukin 6 (IL-6) and leptin induces epithelial to mesenchymal transition (EMT) and CSCsthough JAK/STAT3 signaling. Tumor necrosis factor α (TNFα) induces trans endothelial migration and CSCs though nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. Fatty acid binding proteins (FABP) modulates fatty acid metabolism from neighboring adipocytes to provide fuel for tumor cells to metastasis. Matrix metalloproteinase 11 (MMP-11) dedifferentiates mature adipocytes into cancer associated adipocytes (CAAs) leading to extra cellular matrix (ECM) remodeling and fibrosis. Asparagine promotes EMT in primary tumors and increases tumor cell proliferation at distinct metastatic sites. Inhibiting IL-1R signaling using the receptor antagonist Anakinra resulted in decreased bone metastasis in an in vivo TNBC model. Pretreatment of Anakinra did not prevent tumor cell numbers that arrived to the bone, but instead held the tumor cells in a dormant state, thereby preventing metastasis. Furthermore, therapeutic treatment of bone metastasis in the model with Ankinira stalled tumor growth by reducing proliferation and angiogenesis. Anakinra and other IL-1 signaling pathway inhibitors are currently in multiple clinical trials to target metastasis in solid tumors, multiple myeloma and plasma cell neoplasm, as summarized in. In a clinical trial of patients with smoldering or indolent multiple myeloma at risk of progression to active myeloma, Anakinra treatment decreased myeloma proliferative rate and hs-CRP in responders leading to improved progression free survival. A large cardiovascular randomized control trial of Canakinumab (Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS)), an interleukin (IL)-1β inhibitory antibody, noted a dramatic reduction in the incidence of lung cancer. The trial of 10,500 patients was not designed to study lung cancer as an endpoint and the patients were extensively screened prior to recruitment. These results indicate that IL-1β reduced the progression, invasiveness and metastatic spread of early stage lung cancers that were undiagnosed at the time of recruitment. Therefore these studies suggest that anti IL-1β therapy may prevent the metastatic cascade if given early in the disease process, and further studies are required to examine if metastatic spread is also prevented in people living with obesity. However, there is emerging evidence that anti IL-1β therapy may promote metastatic spread if given later in disease development. Castano et al. eloquently reported that primary tumors elicit a systemic inflammatory response involving IL-1β expressing innate immune cells that infiltrate distant metastasis microenvironments. At the metastatic site, IL-1β maintains metastasis initiating cells in a ZEB-1 positive differentiation state, but inhibition of IL-1 receptor relieves the differentiation block and resulted in metastatic colonization. People living with obesity experience more chemoresistance due to altered drug pharmacokinetics and enhanced proinflammatory and adipokine secretions. Therefore further research is required to investigate if inhibitors of downstream intracellular pathways associated with both obesity and chemo-resistance, such as AKT or NF-κB pathways, are clinically superior in combination with chemotherapy in people with obesity. On the other hand, immune checkpoint inhibitors appear to be more effective in the obese population compared to lean patients. The chronic inflammation and lipid accumulation induced by obesity leads to an "exhausted" T cell phenotype, which is associated with PD-1 upregulation. Obese melanoma patients treated with immune checkpoints inhibitors have an improved overall survival and progression free survival compared to lean patients. In addition, response to PD-1 inhibitors has been modest in TNBC however results from the JAVELIN trial suggests that obese TNBC patients with an exhausted immune response might benefit from checkpoint blockage. However, aged mice with excess adiposity developed a lethal cytokine storm reaction following immunotherapy, potentially indicating that older people with obesity may be at a higher risk of adverse effects. Adipose derived fatty acid binding proteins (FABPs) in the tumor microenvironment have important roles as intracellular lipid chaperones and pharmacological agents that modify FABP function are in development for obesity, diabetes and atherosclerosis. FABP5 is not expressed in the normal prostate but is highly upregulated in metastatic prostate cancer and offers is a novel therapeutic target for pharmaceutical inhibition. However, lipid metabolism is central to the function of regulatory T cells (Tregs). Inhibition of FABP5 in Tregs triggers mitochondrial DNA release, which induces cGAS-STING dependent type 1 interferon signaling and the production of IL-10. Overall this promotes Tregs immunosuppressant activity within the tumor microenvironment leading to enhanced tumor progression. Treg activity is associated with a poor prognosis and therefore the effect of pharmacological inhibition of FABP5 would have to be carefully evaluated in all cells within the tumor microenvironment. CD36 is a transmembrane glycoprotein that mediates lipid uptake and binds to a diverse range of ligands including apoptotic cells, thrombospondin-1 (TSP-1) and fatty acids. High fat diet or palmitic acid enhances a subpopulation of CD36 + cells with metastatic potential in mouse models of human oral cancer. The presence of CD36 + metastasis initiating cells also correlated with poor prognosis in a number of cancer types and anti-CD36 neutralizing antibodies induced regression of the lymph node and lung metastasis in murine models. However, there is also evidence that loss of CD36 in stromal cells and surrounding endothelial cells can increase the metastatic potential of the niche and induce angiogenesis. Furthermore, several agents targeting CD36 in clinical trials with thrombospondin mimetic peptides have been unsuccessful due to adverse effects and limited efficacy. CD36 may be promising to specifically target metastasis however further knowledge of its role in stromal cells and downstream signaling pathways is required to develop more precise therapeutics. As glutamine levels are decreased in obesity and cells adapt to low glutamine by becoming dependent on asparagine, an amino acid, which promotes multiple stages of the metastatic cascade, targeting asparagine may prevent obesity-induced metastasis. Indeed, treatment with L-asparaginase, an enzyme that depletes asparagine, or alternatively dietary asparagine restriction, reduced metastasis in a breast cancer model without affecting the growth of the primary tumor. L-asparagine is a mainstay treatment in childhood acute lymphoblastic leukemia (ALL) and children with obesity have a 50% greater risk of ALL relapse than lean counterparts. Glutamine synthesis was markedly increased in bone marrow adipocytes following chemotherapy and obesity substantially impaired L-asparaginase efficacy due to adipocytes secreting glutamine. In addition, obesity increases the risk of hepatic toxicity with L-asparaginase therapy by promoting a maladaptive integrated stress response. Therefore, whilst inhibiting asparagine preferentially targeted metastasis, the experience of L-asparaginase in ALL suggests that it could be less effective and more toxic in people with obesity. Another proposed method to target the metabolism of tumor cells is through short term dietary fasting. Fasting diets are currently in clinical trial to enhance chemotherapy efficacy and reduce toxicity however there is also evidence they may slow tumor progression. In the fasting state IGF-1, a promoter of EMT, is inhibited, and clinical trials are underway to investigate if tumor progression can be inhibited. Obesity induces lung neutrophilia leading to increased breast cancer lung metastasis through GM-CSF and IL-5 signaling. Interestingly, weight loss reversed this effect, leading to reduced serum GM-CSF and IL-5 in both mouse and humans. In addition, this suggests that the disruption of normal lung homeostasis is critical in obesity induced metastasis and pharmacological interventions that resolve lung inflammation may reduce metastatic risk. Targeting IL-5 was shown to be sufficient to block lung neutrophil trafficking in obese mice and there are a number of anti-IL-5 therapies with FDA approval for severe hypereosinophilic asthma. Therefore, obese cancer patients with increased neutrophilia, GM-CSF or IL-5, may benefit from anti-IL-5 therapies to prevent lung metastasis development. ## Targeting metastasis via the treatment of obesity Complementary treatment of obesity may potentially improve outcomes in cancer patients. Indeed, a systematic review and meta-analysis of animal models concluded that weight loss decreases cancer progression and metastasis. The most common method of weight management is dietary calorie restriction (CR) and increasing energy expenditure though exercise. CR extends lifespan and reduces age related diseases, including cancer, in preclinical models and recently translational studies have tested the potential of CR as an adjuvant therapy in cancer. However, chronic CR is often contra indicated in cancer patients due to the risks of malnutrition and cachexia and other strategies, such as intermittent fasting, CR mimetic drugs or alterative diets (e.g., ketogenic diet) may be more suitable. Clinical studies have shown that both adjuvant and neo-adjuvant chemotherapy is less effective in people with obesity. CR, fasting or fasting mimicking diets (FMDs) may improve response to both chemotherapy and radiotherapy as well asreducing side effects associated with cytotoxic drugs. In addition, obesity is associated with an increase desmoplasia surrounding the tumor tissue, contributing to chemoresistance, and CR can reduce the desmoplasia to facilitate better therapeutic drug delivery to the tumor cells. CR, fasting and FMD also have been shown to reduce secreted factors within the tumor microenvironment including proinflammatory cytokines, leptin and IGF-1. Considering the key roles of these molecules to be dysregulated in obesity and promote metastasis, CR, fasting or FMD may reduce tumor progression through this mechanism. Interestingly, however, a preclinical study of colon cancer showed that an intermittent fasting diet expanded the CSCs population and enhanced EMT by promoting the Warburg/Crabtree effect following post-fasting food overconsumption. Therefore, carefully controlled clinical studies of CR, fasting and FMD are required to determine if they have a role in preventing cancer progression without leading to nutritional deficiencies or cachexia. With this in mind, weight management strategies may be a more appropriate strategy to prevent reoccurrence during long-term cancer survivorship. Physical inactivity is a well-known avoidable risk factor for developing cancer. However, there is also evidence that an increased level of physical activity between before to after diagnosis reduces cancer mortality. In contrast, a decreased physical activity level between pre-to post-diagnosis is associated with higher cancer mortality. Together this indicates that tumor progression could be slowed or prevented by physical activity and indeed physical activity regulates multiple steps of the metastatic cascade, as comprehensively reviewed in. In summary, moderate intensity exercise appears to prevent tumor spread by normalizing angiogenesis, reducing circulating tumor cells and decreasing endothelial cell permeability. On the contrary, high-intensity exercise may favor cancer dissemination and promote the formation of the premetastatic niche through excessive stress. In overweight or obese breast cancer survivors, moderate to vigorous exercise three times a week for 16 weeks reduced systemic levels of insulin, IGF-1 and leptin, and increased adiponectin. Therefore, chronic adaptations to moderate-intensity endurance exercise may be the most effective way to achieve a preventive effect of exercise on metastases. Overall, exercise and nutritional strategies may be especially beneficial in overweight or obese cancer survivors to reduce both obesity-associated comorbidities and prevent cancer reoccurrence. Bariatric surgery is the most effective treatment for obesity and its associated metabolic and cardiovascular complications. On the whole studies report that overall cancer incidence and mortality is reduced post bariatric surgery, particularly in women. Obesity is responsible for increased estradiol production in adipose tissue thus driving hormone sensitive cancers and the augmented concentration of total serum estrogen following bariatric surgery is suggested to be the reason for reduced post-menopausal breast and endometrial cancer risk. Indeed bariatric surgery may also reduce tumor progression by specifically treating hyperinsulinemia and reducing high circulating IGF-1 levels. However, data is conflicting and there is a potential increase in cancer incidence following bariatric surgery. There are reports suggesting bariatric surgery results in an increased risk of colorectal cancer (CRC) but the results are complicated as control groups were not standardized for weight changes, dietary changes and medications, which can have a profound impact on CRC incidence. One hypothesis for the increased CRC incidence is based upon a study investigating colonic epithelial cell proliferation in subjects undergoing roux-en-Y-gastric bypass surgery. Biopsies comparing the day of surgery and 6 months after surgery found an increase in the mitotic activity of colonic cells following surgery and an increased expression of proinflammatory and tumorigenic COX-1 and COX-2 enzymes. Bariatric surgery can successfully achieve weight loss among cancer survivors, but long term effects have not been reported. Overall, current evidence indicates bariatric surgery is more effective for cancer prevention in female hormone driven cancers compared to male cancers. Indeed, a study of women with BMI > 40 kg/m 2 who underwent bariatric surgery resulted in a reduction of endothelial proliferation marker and resolved most cases of the precancerous condition atypical hyperplasia. Randomized control trials are required to determine if bariatric surgery in obese women can increase breast or endometrial cancer survivorship and reduce tumor reoccurrence. # Conclusions Obesity is a growing and significant global health crisis, with epidemiological studies demonstrating a compelling association between cancer risk and progression. A clearer understanding of the molecular processes associated with obesity-induced cancer is warranted and has the potential to uncover new targeting strategies. Indeed, potential new targets are already emerging. It is reasonable to posit that weight loss might be beneficial to reduce metastasis risk. However, it is also possible that weight loss alone is insufficient to reverse the effects of chronic obesity due to potential epigenetic reprogramming of tumor cells and/or stromal cells. In addition, patients with advanced disease often experience WAT loss and wasting (cachexia), which complicates treatment and adversely affects patient survival. The Breast Cancer Weight Loss (BWEL) trial (Identifier NCT02750826) is a phase III randomized trial currently underway assessing the impact of a weight loss intervention on disease recurrence in women with stage II to III HER2-negative breast cancer. The results of this trial will help provide answers to some of these questions. Given the limitations of BMI as a measurement of adiposity, novel methods of identifying people with metabolically unhealthy adipose tissue is required. Additional strategies may also benefit individuals who are not obese (BMI< 30) but who have signs of metabolic syndrome or adipose tissue dysfunction. Ultimately, there is a definitive need for more tailored interventions for obese patients to improve survival status in this patient population, especially considering the higher risk of drug resistance in the obese setting.

Acute exacerbation of unclassifiable idiopathic interstitial pneumonia: comparison with idiopathic pulmonary fibrosis Background: Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is well known as a lifethreatening condition during its clinical course. However, the clinical features and prognosis in AE of unclassifiable idiopathic interstitial pneumonia (AE-UCIIP) remain to be elucidated. The aim of this study was to clarify the clinical features and prognosis of AE-UCIIP compared with those of AE-IPF. Methods: In 187 patients with UCIIP or IPF, 64 patients with AE-UCIIP or AE-IPF, who were diagnosed and treated at our hospital, were retrospectively evaluated. Results: A total of 24 patients with AE-UCIIP were significantly older (p = 0.011), included more women (p < 0.001) and never-smokers (p < 0.001), and showed fewer lung lesions on high-resolution computed tomography (p = 0.006) than 40 patients with AE-IPF. Incidence of AE-UCIIP was 10.29%/year and was significantly higher than in AE-IPF (Gray's test, p = 0.008). Prognosis of AE-UCIIP was as poor as that of AE-IPF (log-rank, p = 0.681). Percent-predicted forced vital capacity (%FVC) [hazard ratio (HR) 0.934, p = 0.045], and GAP stage within 12 months before AE (HR 3.530, p = 0.023), and partial pressure arterial oxygen/fraction of inspired oxygen (PaO 2 /FiO 2 ) ratio at AE (HR 0.998, p = 0.016) were significant prognostic factors. Finally, commencement of long-duration (⩾12 h) direct hemoperfusion with a polymyxin B-immobilised fibre column (PMX-DHP) within 2 days after admission significantly improved survival (logrank, p = 0.038) and was a significant prognostic factor (HR 0.175, p = 0.0039) in AE-UCIIP. Long-duration PMX-DHP showed favourable treatment effects even in the combined group of patients with AE-UCIIP or AE-IPF (log-rank p = 0.002; HR 0.328, p = 0.006). Conclusions: Patients with AE-UCIIP were older and included more women and neversmokers than those with AE-IPF. Prognosis of AE-UCIIP was as poor as that of AE-IPF.The reviews of this paper are available via the supplemental material section.Keywords: acute exacerbation, direct hemoperfusion with a polymyxin B-immobilised fibre column, idiopathic pulmonary fibrosis, idiopathic unclassifiable interstitial pneumonia # Introduction Idiopathic interstitial pneumonias (IIPs) are life-threatening diseases that show a variety of inflammation and fibroses on the lung parenchyma, with several pathological and radiological patterns. IIPs are categorized based on their interstitial pneumonia (IP) patterns, and idiopathic pulmonary fibrosis (IPF) is the most frequent IIP with the poorest prognosis. [bib_ref] ATS/ ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis. An official ATS/ERS/JRS/ ALAT statement:..., Raghu [/bib_ref] [bib_ref] statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias, Travis [/bib_ref] Precise classification of IIPs requires a multidisciplinary approach. However, even with a multidisciplinary approach, many patients with IIPs cannot be given a specific IIP diagnosis and are labelled with unclassifiable idiopathic interstitial pneumonia (UCIIP) in clinical practice. [bib_ref] statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias, Travis [/bib_ref] [bib_ref] Prevalence and prognosis of unclassifiable interstitial lung disease, Ryerson [/bib_ref] [bib_ref] Interstitial lung disease in the elderly, Patterson [/bib_ref] The survival of UCIIP is reportedly intermediate between that of IPF and non-IPF, [bib_ref] Prevalence and prognosis of unclassifiable interstitial lung disease, Ryerson [/bib_ref] [bib_ref] Unclassifiable interstitial lung disease: from phenotyping to possible treatments, Guler [/bib_ref] and effective treatments for UCIIP have not been established. Acute exacerbation (AE) of IP is a devastating condition that frequently occurs in the clinical course of IPF, 1,6-8 and AE-IPF accounts for 30-40% of deaths in patients with IPF. [bib_ref] Clinical course and prediction of survival in idiopathic pulmonary fibrosis, Ley [/bib_ref] [bib_ref] Epidemiologic survey of Japanese patients with idiopathic pulmonary fibrosis and investigation of..., Natsuizaka [/bib_ref] Furthermore, AE occurs not only in IPF but also in other IIPs such as non-specific interstitial pneumonia or connective tissue disease-associated IP. [bib_ref] Acute exacerbation of interstitial pneumonia other than idiopathic pulmonary fibrosis, Park [/bib_ref] [bib_ref] Acute exacerbation of interstitial pneumonia associated with collagen vascular diseases, Suda [/bib_ref] [bib_ref] Differences in clinical features of acute exacerbation between connective tissue diseaseassociated interstitial..., Enomoto [/bib_ref] However, little is known about AE in patients with UCIIP, while we sometimes experience AE-UCIIP in clinical practice. Here, in the present study, we retrospectively evaluated clinical features of AE-UCIIP compared with those of AE-IPF. Furthermore, the prognostic factors including treatments were examined in patient with AE-UCIIP. To our knowledge, this is the first study to provide detailed information about clinical features and prognosis in patients with AE-UCIIP. # Methods ## Study design and subjects Among 187 patients with UCIIP or IPF, 77 patients who were diagnosed and treated for AE-UCIIP or AE-IPF at our hospital between 2002 and 2018 were retrospectively reviewed. The diagnosis of UCIIP was re-evaluated based on the American Thoracic Society (ATS) and European Respiratory Society (ERS) international statement for classification of IIPs in 2013. [bib_ref] statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias, Travis [/bib_ref] The diagnosis of IPF was also re-evaluated based on the official clinical practice guideline of the ATS, ERS, Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT) in 2018. [bib_ref] Diagnosis of idiopathic pulmonary fibrosis. An official ATS/ ERS/JRS/ALAT clinical practice guideline, Raghu [/bib_ref] Patients with AE-IP who had an acute, clinically significant respiratory deterioration characterized by evidence of new widespread alveolar abnormality were selected. All patients met the modified diagnostic criteria described by Briefly, the diagnostic criteria for AE-IP were as follows: (a) previous or concurrent diagnosis of IP; (b) acute worsening or development of dyspnoea typically <1 month in duration; (c) computed tomography (CT) with new bilateral ground-glass opacity and/or consolidation superimposed on background findings of IP; and (d) deterioration not fully explained by cardiac failure or fluid overload. A total of 11 patients with a second recurrence of AE-IP and 1 patient with second and third recurrence of AE-IP were excluded so that the prognosis was evaluated from the first AE-IP in 64 patients with AE-UCIIP or AE-IPF (Supplemental [fig_ref] Figure 1: Figure 1 [/fig_ref]. The study protocol was approved by the Ethics Committee of Hamamatsu University School of Medicine (approval number 18-085). All procedures in this study were performed in accordance with the study protocol and the 1964 Declaration of Helsinki as amended. The need for patient approval and informed consent was waived due to the retrospective nature of the study. Incidence of first acute exacerbation (AE) and Kaplan-Meier survival curves from the first AE onset. Incidence of first AE in patients with unclassifiable idiopathic interstitial pneumonia (AE-UCIIP) was significantly higher than that in AE in patients with idiopathic pulmonary fibrosis (AE-IPF) (A, Gray's test, p = 0.008). The 12-month survival rate in patients with AE-UCIIP was as poor as those with AE-IPF (B, log-rank test, p = 0.681). journals.sagepub.com/home/tar 3 ## Data collection Clinical, laboratory, and physiological data were obtained from medical records. Disease severity of IP within 12 months before the AE event was assessed using the gender, age, and physiology (GAP) staging system 16 and the Japanese Respiratory Society (JRS) severity grades for IP. [bib_ref] Usefulness of a disease severity staging classification system for IPF in Japan:..., Homma [/bib_ref] The former considers gender, age, and two lung physiology variables: forced vital capacity (FVC) and diffusion lung capacity for carbon monoxide (DL CO ). [bib_ref] A multidimensional index and staging system for idiopathic pulmonary fibrosis, Ley [/bib_ref] The latter consists of partial pressure arterial oxygen (PaO 2 ) at rest and minimum oxygen saturation (SpO 2 ) during the 6-minute walking test (6MWT). [bib_ref] Usefulness of a disease severity staging classification system for IPF in Japan:..., Homma [/bib_ref] The extent of lung opacity was measured on three high-resolution CT (HRCT) slices as described in our previous report. [bib_ref] Amount of elastic fibers predicts prognosis of idiopathic pulmonary fibrosis, Enomoto [/bib_ref] The pattern of AE-IP on HRCT was classified as (a) peripheral, (b) multifocal, or (c) diffuse, as reported by Akira et al. [bib_ref] Computed tomography findings in acute exacerbation of idiopathic pulmonary fibrosis, Akira [/bib_ref] The HRCT findings were reviewed by two observers. ## Pmx-dhp therapy # Statistical analysis Statistical analysis was performed using JMP-13.1.0 (SAS Institute Inc., Cary, NC, USA) and EZR 1.41 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). [bib_ref] Investigation of the freely available easy-to-use software 'EZR' for medical statistics, Kanda [/bib_ref] Categorical data were compared using the χ 2 test or Fisher's exact probability test for independence, and continuous data using the Wilcoxon rank sum test. AE-IIPs occurrence was estimated considering the death before AE as a competing event, and analysed using Gray's method. Overall survival of patient groups was estimated using Kaplan-Meier curves. The relationships between variables and mortality were evaluated by Cox proportional hazards regression analysis. All tests were two-sided and statistical significance was set at p < 0.05. [fig_ref] Figure 1: Figure 1 [/fig_ref]. Cumulative incidence of AE-UCIIP was 38.1% (24 of 63 patients) and frequency of AE was significantly higher in AE-UCIIP than in AE-IPF (Gray's test, p = 0.008). Further, the median period from IP diagnosis to first AE was significantly shorter in patients with UCIIP than in those with IPF [fig_ref] Table 1: Comparison of data between patients with AE-IPF and AE-UCIIP [/fig_ref] 14.5 versus 74.5 months, p < 0.001). # Results ## Incidence of acute exacerbation in patients with ## Clinical characteristics, physiological examination findings, and treatments: comparison between patients with ae-uciip and ae-ipf Clinical characteristics of all patients with AE-UCIIP, including physiological examination findings and treatments, are shown in [fig_ref] Table 1: Comparison of data between patients with AE-IPF and AE-UCIIP [/fig_ref] and compared with patients with AE-IPF. In 24 patients with AE-UCIIP, 7 were diagnosed with UCIIP after surgical lung biopsy (SLB). Patients with AE-UCIIP had a median age of 79.5 years at the diagnosis of AE-UCIIP and were significantly older than those with AE-IPF (69.5 years old, p = 0.011). More female patients with AE-UCIIP (p < 0.001) and more never-smokers (p < 0.001) were found in comparison with patients with AE-IPF. The period from first IP diagnosis to first AE in AE-UCIIP was significantly shorter than in AE-IPF (74.5 versus 14.5 months, p < 0.001). As for lung function within 12 months before AE, %FVC tended to be higher in AE-UCIIP than in AE-IPF (62.0 versus 55.7%, p = 0.051). %DL CO was not different (p = 0.414). The extent score of lung fibrosis within 12 months before AE was significantly lower in AE-UCIIP than in IPF (p = 0.006). Severity scores of interstitial pneumonia within 12 months before AE (GAP and JRS scores) were not different (p = 0.555 and 0.132, respectively). The proportion of patients receiving previous treatments, including steroids, immunosuppressants, and oxygen therapy, was not different between groups. At AE, serum markers were not different between the groups. The partial pressure arterial oxygen/fraction of inspired oxygen (PaO 2 /FiO 2 ) (P/F) ratio, extent score of all HRCT findings, and HRCT pattern at AE were not different either. All patients were treated with steroid-pulse therapy after hospital admission (methylprednisolone at 1000 mg/day for 3 days) followed by a tapering dose of prednisolone. Treatments with an immunosuppressant (cyclophosphamide or cyclosporine) and/or longduration (mainly ⩾12 h) PMX-DHP 20,22 were added to steroid therapy in more than half of patients. A total of 2 in 24 patients with AE-UCIIP and 2 in 40 patients with AE-IPF had concomitant respiratory infection at the time of admission (triggered AE). Serum endotoxin was undetectable in all patients treated with PMX-DHP. These treatments were commenced concomitantly with the antibiotics as soon as possible after admission. None of these AE treatments were different between groups. ## Mortality rate and prognostic factors in patients with ae-uciip Of 24 patients with AE-UCIIP, 4 died within 1 month of onset (mortality rate, 16.7%), 10 died within 3 months (mortality rate, 41.7%), and 13 died within 12 months (mortality rate, 54.2%). Within 12 months of AE-UCIIP onset, 10 patients died of respiratory failure, 1 died of bacterial pneumonia, and 2 died of lethal arrythmia. [fig_ref] Figure 1: Figure 1 [/fig_ref] shows Kaplan-Meier survival curves from the first AE onset. The 12-month survival rate in patients with AE-UCIIP was as poor as those with AE-IPF (log-rank test, p = 0.681). The results of univariate Cox proportional hazards models of survival in AE-UCIIP to identify prognostic factors are shown in [fig_ref] Table 2: Univariate Cox proportional hazards models of survival in patients with AE-UCIIP [/fig_ref]. %FVC within 12 months before AE was a significant prognostic factor in patients with AE-UCIIP [hazard ratio (HR) 0.928, p = 0.025]. Similarly, the GAP stage within 12 months before AE was also a significant prognostic factor (HR 4.467, p = 0.005). At AE, the P/F ratio was significant (HR 0.988, p = 0.005), and peripheral blood white blood cells (WBC) and serum lactate dehydrogenase (LDH) tended to be a prognostic factor (HR 1.000, p = 0.095 and HR 1.002, p = 0.097, respectively). All patients were treated with steroid-pulse therapy. In addition to this, 14 patients with AE-UCIIP were treated with long-duration PMX-DHP, but this could not improve the survival [fig_ref] Figure 2: Kaplan-Meier survival curves in patients treated with or without long-duration direct hemoperfusion... [/fig_ref] , p = 0.508). However, in AE-UCIIP patients treated within 2 days after admission, commencement of long-duration PMX-DHP improved survival [fig_ref] Figure 2: Kaplan-Meier survival curves in patients treated with or without long-duration direct hemoperfusion... [/fig_ref] ## Mortality rate and prognostic factors in all patients with ae-uciip or ae-ipf The 12-month survival rates of AE-UCIIP and AE-IPF patients were almost the same, as shown in [fig_ref] Figure 1: Figure 1 [/fig_ref]. [fig_ref] Figure 2: Kaplan-Meier survival curves in patients treated with or without long-duration direct hemoperfusion... [/fig_ref] , p = 0.029) and was a significantly better prognostic factor (Supplemental [fig_ref] Table 1: Comparison of data between patients with AE-IPF and AE-UCIIP [/fig_ref] , HR 0.505, p = 0.031). Further, in patients treated within 2 days after admission, commencement of longduration PMX-DHP further improved the survival, especially at 3 months after the onset of AE [fig_ref] Figure 2: Kaplan-Meier survival curves in patients treated with or without long-duration direct hemoperfusion... [/fig_ref] , p = 0.002; Supplemental [fig_ref] Table 1: Comparison of data between patients with AE-IPF and AE-UCIIP [/fig_ref] , HR 0.249, p < 0.001). Supplemental [fig_ref] Table 2: Univariate Cox proportional hazards models of survival in patients with AE-UCIIP [/fig_ref] compares data between patients treated with long-duration PMX-DHP and those without the treatment. Although patients receiving longduration PMX-DHP were significantly younger (70 versus 80 years old, p = 0.008) and receiving less previous oxygen therapy (p = 0.048), their %FVC, %DL CO , JRS severity grade, GAP stage within 12 months before AE, and P/F ratio at AE were not different. Next, multivariate Cox proportional hazards models of survival adjusted for GAP stage within 12 months before AE and P/F ratio at AE are shown in [fig_ref] Table 3: Multivariate [/fig_ref]. Extent score on HRCT within 12 months before AE (HR 1.176, p = 0.007) and ΔP/F ratio and ΔLDH at 2 days after beginning AE treatment (HR 0.994, p = 0.048 and HR 1.004, p = 0.024, respectively) were still significant factors. Whereas treatment with long-duration PMX-DHP itself was not significant (HR 0.561, p = 0.124), commencement of long-duration PMX-DHP within 2 days after admission was still a significantly better prognostic factor (HR 0.328, p = 0.006) in patients with AE-UCIIP or AE-IPF. # Discussion In this study, we retrospectively evaluated 24 patients with AE-UCIIP and compared their clinical features and prognosis with those of patients with AE-IPF. The incidence of AE-UCIIP was higher and patients with AE-UCIIP were older, included more women and never-smokers, and showed fewer lung lesions on HRCT than those with AE-IPF. Regarding prognostic factors, %FVC and GAP stage within 12 months before AE, and P/F ratio at AE were significant factors. Finally, commencement of long-duration PMX-DHP within 2 days after admission was a significant prognostic factor, not only in AE-UCIIP but also in the combined group of 64 patients with AE-UCIIP or AE-IPF. To our knowledge, this is the first report to provide detailed information about clinical features and prognosis in patients with AE-UCIIP in comparison to those with AE-IPF. The prevalence of UCIIP has been reported to be as high as 11.9% (0-59.4%).Therefore, a significant number of patients with IIPs are classified as having UCIIPs, and follow-up and treatment of these patients are important in clinical practice. There are three major reasons for UCIIP: (a) incomplete evaluation due to lack of data; (b) presence of overlapping findings that are common to multiple distinct interstitial lung diseases (ILD) subtypes; and (c) non-specific findings that are not characteristic of any single ILD subtype. [bib_ref] Unclassifiable interstitial lung disease: from phenotyping to possible treatments, Guler [/bib_ref] Among these reasons, incomplete evaluation due to a lack of data is the most frequent, and patients with a high risk of SLB, such as old age and low pulmonary function, accounted for 41-52% of the lack of data. [bib_ref] Prevalence and prognosis of unclassifiable interstitial lung disease, Ryerson [/bib_ref] [bib_ref] Unclassifiable interstitial lung diseases: clinical journals.sagepub.com/home/tar characteristics and survival, Hyldgaard [/bib_ref] Even in the current study, patients with AE-UCIIP were significantly older than those with IPF, and only seven patients among 24 could undergo SLB before AE. Regarding AE, the occurrence of AE is an apparent poor prognostic factor in IPF. However, the incidence and significance of AE on the prognosis of UCIIPs is poorly known. Nakamura et al. reported that AE cumulatively occurred in 30.3% of patients with UCIIP. [bib_ref] Clinicoradio-pathological characteristics of unclassifiable idiopathic interstitial pneumonias, Nakamura [/bib_ref] Similarly, in the current study, the cumulative incidence of AE-UCIIP was 38.1% and the frequency of AE was significantly higher (10.29%/year) in AE-UCIIP than in AE-IPF. Further, the survival of patients with AE-UCIIP was as poor as that of AE-IPF. Therefore, great attention should be paid to prevent AE and to start early treatments against AE-UCIIP. As for the mechanism of AE-IP, infiltration of activated neutrophils into the lung parenchyma in the early phase of AE plays a pivotal role, [bib_ref] Acute exacerbation of idiopathic pulmonary fibrosis. An international working group report, Collard [/bib_ref] and early removal of these neutrophils seems to be important for saving patients with AE-IP, which does not have established treatments. Basically, PMX-DHP was developed to remove endotoxins from patients with sepsis, but it could also remove activated neutrophils in AE-IPF patients. [bib_ref] Neutrophil adsorption by polymyxin B-immobilized fiber column for acute exacerbation in patients..., Abe [/bib_ref] Furthermore, removal of several mediators by PMX-DHP, which can facilitate vascular permeability, may also have a role for the treatment of AE-IP. Treatment with PMX-DHP reportedly decreased the concentrations of serum matrix metalloproteinase-9, 26 vascular endothelial growth factor, [bib_ref] Association between cytokine removal by polymyxin B hemoperfusion and improved pulmonary oxygenation..., Oishi [/bib_ref] and angiopoietin-2. [bib_ref] Treatment of acute exacerbation of idiopathic pulmonary fibrosis with direct hemoperfusion using..., Enomoto [/bib_ref] [bib_ref] Angiopoietin balance in septic shock patients treated by direct hemoperfusion with polymyxin..., Ebihara [/bib_ref] [bib_ref] Angiopoietin balance in septic shock patients with acute lung injury: effect of..., Ebihara [/bib_ref] Regarding the performing time of PMX-DHP, long-duration PMX-DHP significantly improved survival in patients with AE-IPF. [bib_ref] Treatment of acute exacerbation of idiopathic pulmonary fibrosis with direct hemoperfusion using..., Enomoto [/bib_ref] [bib_ref] Possible therapeutic effect of direct haemoperfusion with a polymyxin B immobilized fibre..., Enomoto [/bib_ref] [bib_ref] Polymyxin B-immobilized fiber column (PMX) treatment for idiopathic pulmonary fibrosis with acute..., Abe [/bib_ref] [bib_ref] Evaluation of different perfusion durations in direct hemoperfusion with polymyxin B-immobilized fiber..., Kono [/bib_ref] In addition, early treatment with long-duration PMX-DHP was effective for improving survival of AE-IPF patients compared with later commencement of PMX-DHP. [bib_ref] Survival from an acute exacerbation of idiopathic pulmonary fibrosis with or without..., Oishi [/bib_ref] [bib_ref] Direct hemoperfusion with polymyxin B-immobilized fibre treatment for acute exacerbation of interstitial..., Furusawa [/bib_ref] In the current study, commencement of long-duration PMX-DHP within 2 days after admission could also improve survival and was a significant prognostic factor both in patients with AE-UCIIP and all patients with AE-UCIIP or AE-IPF. Aggressive and early treatment with long-duration PMX-DHP may further improve the prognosis especially in patients with AE-UCIIP who had fewer lung lesions on HRCT than those with AE-IPF. As for infection, the current definition of AE-IPF admits 'triggered AE' including infection in addition to 'idiopathic AE'. [bib_ref] Acute exacerbation of idiopathic pulmonary fibrosis. An international working group report, Collard [/bib_ref] In this setting, even in the presence of concomitant infection, PMX-DHP may benefit treatment of AE-IP because of its original capacity to remove serum endotoxin by sepsis, although serum endotoxin was undetectable in all patients treated with PMX-DHP in this study. This study has several limitations. First, only a small number of patients with AE-UCIIP were included. Second, the data were retrospectively collected. Patients with connective tissue diseaserelated IP were excluded in this study; therefore, those with UCIIP preceding connective tissue diseases may have been missed. Third, in patients with AE-UCIIP, the potential selection bias exists that diagnosis of IPF may have been missed because old UCIIP patients were less likely to be referred for SLB. However, many such old UCIIP patients, who cannot undergo SLB due to a high risk of biopsy, reportedly exist in real-world clinical practice. [bib_ref] Prevalence and prognosis of unclassifiable interstitial lung disease, Ryerson [/bib_ref] [bib_ref] Unclassifiable interstitial lung diseases: clinical journals.sagepub.com/home/tar characteristics and survival, Hyldgaard [/bib_ref] Fourth, prognostic factors in AE-UCIIP could not have been compared with those in AE-IPF using multivariate analysis due to a small number of patients. Finally, the treatment for AE-UCIIP was not uniform. Therefore, a larger prospective study is needed precisely to assess the clinical features and treatments for AE-UCIIP. # Conclusion We retrospectively studied 24 patients with AE-UCIIP and 40 patients with AE-IPF. The frequency of AE was higher in AE-UCIIP than in AE-IPF. Patients with AE-UCIIP were older and included more women and never-smokers than those with AE-IPF. %FVC and GAP stage within 12 months before AE, and P/F ratio at AE were significant prognostic factors. Although the prognosis of AE-UCIIP was as poor as that of AE-IPF, commencement of long-duration PMX-DHP therapy within 2 days after admission improved survival not only in AE-UCIIP patients but also in AE-IPF patients. These findings should be useful for the management and treatment of patients with AE-UCIIP in clinical practice. Further evaluation is needed to improve the poor survival of patients with AE-UCIIP. [fig] Figure 1: Figure 1. Incidence of first acute exacerbation (AE) and Kaplan-Meier survival curves from the first AE onset. Incidence of first AE in patients with unclassifiable idiopathic interstitial pneumonia (AE-UCIIP) was significantly higher than that in AE in patients with idiopathic pulmonary fibrosis (AE-IPF) (A, Gray's test, p = 0.008). The 12-month survival rate in patients with AE-UCIIP was as poor as those with AE-IPF (B, log-rank test, p = 0.681). [/fig] [fig] Figure 2: Kaplan-Meier survival curves in patients treated with or without long-duration direct hemoperfusion with a polymyxin B-immobilised fibre column (PMX-DHP). All of the patients were treated with steroid-pulse therapy. In addition to this, 14 out of 24 patients with acute exacerbation of unclassifiable idiopathic interstitial pneumonia (AE-UCIIP) were treated with long-duration PMX-DHP. Long-duration PMX-DHP treatment could not improve the survival (A, log-rank, p = 0.508). In AE-UCIIP patients treated within 2 days after admission, long-duration PMX-DHP significantly improved survival (B, log-rank, p = 0.038). In all 64 patients with AE-UCIIP or idiopathic pulmonary fibrosis (AE-IPF), treatment with long-duration PMX-DHP improved survival (C, log-rank, p = 0.029). In these patients treated within 2 days after admission, long-duration PMX-DHP further improved the survival, especially at 3 months after the onset of AE (D, log-rank, p = 0.002).8 journals.sagepub.com/home/tar PMX-DHP improved survival ( [/fig] [table] Table 1: Comparison of data between patients with AE-IPF and AE-UCIIP. [/table] [table] Table 2: Univariate Cox proportional hazards models of survival in patients with AE-UCIIP (n = 24). [/table] [table] Table 3: Multivariate [/table]

Accurate Sparse-Projection Image Reconstruction via Nonlocal TV Regularization Sparse-projection image reconstruction is a useful approach to lower the radiation dose; however, the incompleteness of projection data will cause degeneration of imaging quality. As a typical compressive sensing method, total variation has obtained great attention on this problem. Suffering from the theoretical imperfection, total variation will produce blocky effect on smooth regions and blur edges. To overcome this problem, in this paper, we introduce the nonlocal total variation into sparse-projection image reconstruction and formulate the minimization problem with new nonlocal total variation norm. The qualitative and quantitative analyses of numerical as well as clinical results demonstrate the validity of the proposed method. Comparing to other existing methods, our method more efficiently suppresses artifacts caused by low-rank reconstruction and reserves structure information better. # Introduction Computed tomography (CT) has still been a widely used medical imaging technology for clinical diagnosis. However, according to the recent reports, the risk of overhigh radiation has caused social attention. It is well known that it is harmful for human body to expose to heavy radioactive source. As a result, the problem which arises is, when CT scans are inevitable, and how can we reduce the radiation dose without losing the imaging quality? To deal with this issue, many technologies which can be categorized into two groups were proposed. The first one is to lower the configuration parameters of X-ray. The key step is to reduce the milliampere seconds (mAs) or kVp parameter; however, the quantum noises also appear. Many methods were proposed to suppress the quantum noises [bib_ref] Nonlinear sinogram smoothing for low-dose X-ray CT, Li [/bib_ref] [bib_ref] Electronic noise modeling in statistical iterative reconstruction, Xu [/bib_ref] [bib_ref] Low-dose computed tomography image restoration using previous normal-dose scan, Ma [/bib_ref] [bib_ref] Noise reduction in computed tomography scans using 3-D anisotropic hybrid diffusion with..., Mendrik [/bib_ref] [bib_ref] Improving low-dose abdominal CT images by weighted intensity averaging over large-scale neighborhoods, Chen [/bib_ref] [bib_ref] Statistical sinogram smoothing for low-dose CT with segmentation-based adaptive filtering, Zhang [/bib_ref]. The vital problem of this kind is that under the situation of low operational current or voltage, when high density objects, such as metal implants or bones exit, the severe attenuation of X-rays allows only a limited number of photons to reach detectors. As a result, new artifacts will be introduced in the reconstructed image. The artifacts spread through the whole image, which contaminate the imaging quality. Therefore, the second category does not change the energy of X-ray. Instead of that, reducing the projection number which is also called sparse-projection reconstruction is another way to achieve this goal. However, due to the lack of projection views, streak artifacts will severely affect the imaging quality. This topic can be viewed as an ill-posed inverse problem which has provoked many studies about it. In this paper, we will focus on sparse-projection reconstruction. Compressive sensing (CS) is an efficient method to handle sparse-projection reconstruction [bib_ref] Robust uncertainty principles: exact signal reconstruction from highly incomplete frequency information, Candès [/bib_ref] [bib_ref] Compressed sensing, Donoho [/bib_ref]. The main idea of CS is very similar to sparse-projection reconstruction and both of them manage to recover the complete signals from a severe undersampling. Many studies have been done following such concepts. In [bib_ref] Prior image constrained compressed sensing (PICCS): a method to accurately reconstruct dynamic..., Chen [/bib_ref] , Chen et al. considered a prior image as a prior knowledge (PICCS). Based on the fact that in many CT imaging applications some physical and anatomical structures and the corresponding attenuation information of the scanned object can be a priori known, Rashed and Kudo presented a statistical iterative reconstruction (SIR) by incorporating this prior information into the image reconstruction objective function [bib_ref] Statistical image reconstruction from limited projection data with intensity priors, Rashed [/bib_ref]. Ma et al. introduced nonlocal means into low-dose reconstruction with a precontrast scan [bib_ref] Iterative image reconstruction for cerebral perfusion CT using a pre-contrast scan induced..., Ma [/bib_ref]. The most famous model with CS theory is total variation (TV) based method called ASD-POCS which is firstly proposed by Sidky et al. [bib_ref] Accurate image reconstruction from few-views and limited-angle data in divergent-beam CT, Sidky [/bib_ref] [bib_ref] Image reconstruction in circular conebeam computed tomography by constrained, total-variation minimization, Sidky [/bib_ref]. They introduced TV into algebraic 2 The Scientific World Journal reconstruction technique (ART) to suppress the artifacts caused by the limitation of projection views. Following this idea, the same group replaced 1 norm with norm in the minimization function [bib_ref] Image reconstruction from few views by non-convex optimization, Sidky [/bib_ref]. Ritschl et al. proposed a step-size-adaptive method based on TV to eliminate the dependence on the raw data consistency [bib_ref] Improved total variation-based CT image reconstruction applied to clinical data, Ritschl [/bib_ref]. To improve the convergence and efficiency of TV based minimization methods, Yu and Wang constructed a pseudoinverse of discrete gradient transform (DGT) and adapted a softthreshold filtering algorithm [bib_ref] A soft-threshold filtering approach for reconstruction from a limited number of projections, Yu [/bib_ref]. Lu et al. proposed a novel algorithm for image reconstruction from few-view data. It utilizes the simultaneous algebraic reconstruction technique (SART) coupled with dictionary learning, sparse representation, and TV minimization on two interconnected levels [bib_ref] Few-view image reconstruction with dual dictionaries, Lu [/bib_ref]. Although TV based methods have achieved efficient results, TV suffered from the notorious blocky effect which obstructs the clinical practice of TV. To overcome this disadvantage; many efforts were made [bib_ref] Anisotropic total variation minimization method for limited-angle CT reconstruction, Jin [/bib_ref] [bib_ref] Image reconstruction from a small number of projections, Herman [/bib_ref] [bib_ref] X-Ray CT image reconstruction via wavelet frame based regularization and radon domain..., Dong [/bib_ref]. Zhang et al. combined classical TV with a high-order norm to suppress the blocky effect [bib_ref] Few-view image reconstruction combining totaln variation and a high-order norm, Zhang [/bib_ref]. Fractional calculus was introduced into ASD-POCS model [bib_ref] Few-view image reconstruction with fractional-order total variation, Zhang [/bib_ref]. By adjusting the order of fractional-order TV norm, blocky effect can be reduced to an acceptable level without increasing much computational cost. The reason of this side effect should put the blame on the basic assumption of TV that the images are piecewise constant and TV is a local-related computation. In this paper, we propose a nonlocal TV based model to deal with the sparse-projection image reconstruction. First, we will review the classical TV based model in the next section. The details of our method are represented in Section 3. Numerical and clinical experiment results are demonstrated in Section 4. The discussion and conclusion are given at the end of this paper. ## A brief review about tv based image reconstruction method Because our method is an extended version of TV based image reconstruction, in this section, we first give a brief description of this method. Given a 2-dimensional image [formula] = (x) = 1 , 2 , whose size is × , 1 ∈ [1, ] and ∇ = (Δ 1 , Δ 2 ) ,(1) [/formula] where Δ 1 and Δ 2 are the first-order differential operators along -axis and -axis, respectively. Δ 1 and Δ 2 can be represented as [formula] Δ 1 = 1 , 2 − 1 −1, 2 , Δ 2 = 1 , 2 − 1 , 2 −1 .(2) [/formula] In traditional CT imaging problem, the sampling procedure can be considered as a discrete linear transform [formula] = ,(3) [/formula] where is the system matrix which is comprised of row vectors and = ( 1 , 2 , . . . , ) is the measurement vector. The individual elements of the system matrix are , where = 1, 2, . . . , and = 1, 2, . . . , . It is obvious that × = . Without losing generality, the fan-beam projection geometry can be demonstrated in [fig_ref] Figure 1: Fan-beam CT geometry configuration [/fig_ref]. To solve the linear system in (3), the TV based image reconstruction algorithm which was used to deal with the sparse-projection limitation is to optimize the following problem [bib_ref] Accurate image reconstruction from few-views and limited-angle data in divergent-beam CT, Sidky [/bib_ref] [bib_ref] Image reconstruction in circular conebeam computed tomography by constrained, total-variation minimization, Sidky [/bib_ref] : [formula] min ‖ ‖ TV subject to ≥ 0, = ,(4) [/formula] where ‖ ‖ TV can be considered as a 1 norm of the firstorder gradient image ∇ . The TV based algorithm combined the steepest decent method and the projection on convex sets (POCS) to achieve the solution of (4) iteratively. ## The proposed nonlocal tv reconstruction method The locality of TV is the main factor which causes blocky effect and it is the motivation we introduce nonlocal TV based method for alleviating this phenomenon. The minimization problem of CT image reconstruction can be formulated as [formula] min ( ) subject to ≥ 0, = ,(5) [/formula] where ( ) is the regularization term and other symbols are with same meanings as [bib_ref] Noise reduction in computed tomography scans using 3-D anisotropic hybrid diffusion with..., Mendrik [/bib_ref]. The key part is how to choose ( ). In TV based model, ( ) = TV ( ) = ‖ ‖ TV and in our proposed method, ( ) = NLTV ( ) = ‖ ‖ NLTV . Inspired by [bib_ref] Nonlocal operators with applications to image processing, Gilboa [/bib_ref] [bib_ref] Nonlocal linear image regularization and supervised segmentation, Gilboa [/bib_ref] [bib_ref] Image recovery via nonlocal operators, Lou [/bib_ref] [bib_ref] Bregmanized nonlocal regularization for deconvolution and sparse reconstruction, Zhang [/bib_ref] , we define ‖ ‖ NLTV as [formula] ‖ ‖ NLTV = ∫ Ω ∇ NLTV (x) x,(6) [/formula] where the nonlocal gradient ∇ NLTV (x) is defined as the vector of all partial differences ∇ NLTV (x, ⋅) at x such that [formula] ∇ NLTV (x, y) = ( (y) − (x)) √ (x, y), ∀y ∈ Ω.(7) [/formula] The Scientific World Journal 3 So we obtain the minimization function [formula] min ∫ Ω √∫ Ω ( (x) − (y)) 2 (x, y) y x, subject to ≥ 0, = ,(8) [/formula] where (x, y) is a weighting function to compute the similarity between vectors x and y. (x, y) is defined as [bib_ref] Image recovery via nonlocal operators, Lou [/bib_ref] ( [formula] x, y) = exp (− * ( (x)) − ( (y)) 2 ℎ 2 ) ,(9) [/formula] where is the Gaussian kernel with standard deviation and ℎ is filtering parameter controlling the decay of the exponential function. Generally, ℎ is determined by the noise level. (x) and (y) denote the two local similarity neighborhoods (named patch windows) centered at the pixels x and y, respectively, and we only compute the weights in a semilocal searching window for each pixel. To simplify the optimization problem, we reformulate (5) as [formula] ( ) = ∫ Ω ∇ NLTV (x) x + 2 − 2 2 ,(10) [/formula] where is a parameter to control the balance between regularization and fidelity terms. Then, we use the gradient descent to update the solution by the Euler-Lagrange equation of (10): [formula] = − + * ( − ) ,(11) [/formula] where * is the adjoint of and [formula] = − ∫ Ω ( (y) − (x)) (x, y) × [ 1 ∇ NLTV (x) + 1 ∇ NLTV (y) ] y.(12) [/formula] Our proposed method for sparse-projection image reconstruction can be summarized by the following steps: (c) inner loop for = 1, 2, . . . , : NLTV gradient descent method: [formula] +1 = − + * ( − ) ;(14) [/formula] (d) repeat beginning with step (b) until the stopping criteria are satisfied. ## Numerical and clinical results In this section, to validate and evaluate the proposed method, numerical and clinical experiments are performed. In the numerical experiments, Shepp-Logan phantom was used and we simulated X-ray projections using Siddon's ray-driven algorithm [bib_ref] Fast calculation of the exact radiological path for a three-dimensional CT array, Siddon [/bib_ref] in fan-beam geometry. The source to rotation center distance is 40 cm and the detector to rotation center is 40 cm. The image array is 20 × 20 cm 2 . The detector whose length is 41.3 cm is modeled as a straight-line array of 512 detector bins. All the tests are performed by MATLAB on a PC with Intel i7-3770 CPU 3.40 GHz and 8 Gb RAM. In clinical experiment, we applied our method to a typical CT slice of a human chest. All the scans were performed on a Siemens SOMATOM Sensation 64 MSCT scanner (Siemens Medical System, Erlangen, Germany) except for the Shepp-Logan (S-L) phantom. The voltage and current were 120 kVp and 200 mA with a slice thickness of 1 mm. To get a good visual effect of our method, we compare the proposed method to FBP, EM, and ASD-POCS. The reconstruction results are also quantitatively evaluated in terms of RMSE and MSSIM whose computational definitions are given in [bib_ref] A novel noniterative metal artifact reduction method using coherence transport with fast..., Zhang [/bib_ref] [bib_ref] Image quality assessment: from error visibility to structural similarity, Wang [/bib_ref]. In all the experiment results, the main parameters were set as = 1, the search window size = 5 × 5, the patch window size = 21 × 21, = 0.1, = 100, and = 20. Moreover, we chose ℎ to be the estimated noise variance in the filtered back projection image. We used a wavelet based noise estimation model introduced by Donoho and Johnstone in these experiments [bib_ref] Ideal spatial adaptation by wavelet shrinkage, Donoho [/bib_ref]. ## Phantom cases. In this section, the numerical experiment results are given. The results were performed under ideal condition, in which projection data were generated numerically without adding noise. To demonstrate the performance of our method, Shepp-Logan phantom was uniformly sampled with 30 over 360 degrees. The iteration numbers of EM, ASD-POCS, and NLTV were simply set to 100. The reconstruction results of Shepp-Logan phantom are given in. In, it is obvious that, due to incompleteness of projection data, classical FBP cannot achieve an acceptable solution. Although EM is a widely used method, its result inalso blurred. Compared to ASD-POCS, NLTV can reduce the impact to a certain extent. The edges inare well kept and three oval organs in the bottom of image are more distinguishable. Meanwhile, the corresponding quantitative evaluations are shown in [fig_ref] Table 1: Evaluations of numerical phantom reconstruction [/fig_ref]. Statistically, NLTV yields better RMSE and MSSIM than those of other methods, but the computational cost is much higher. ## Clinical cases. In this section, we validated the proposed method on a clinical case which was scanned by a Siemens SOMATOM Sensation 64 MSCT scanner (Siemens Medical System, Erlangen, Germany). The voltage and current were 120 kVp and 200 mA with a slice thickness of 1 mm. To demonstrate the effectiveness of our method, the results processed by FBP, EM, and ASD-POCS are given for comparison. The full scanned image with 550 views is used as a reference image. The image is downsampled uniformly to 20 views, about two-tenths of the original dataset. The iteration numbers of EM, ASD-POCS, and NLTV were set to 100 uniformly. The reconstructed images are displayed in [fig_ref] Figure 3: Reconstruction results of chest image by different methods [/fig_ref]. It is obvious that FBP and EM cause considerable streaklike artifacts in [fig_ref] Figure 3: Reconstruction results of chest image by different methods [/fig_ref] and 3(c) and the structure information of tissues is of terrible vision. ASD-POCS and NLTV dispel most of the artifacts, so that most of the tissues can be seen in [fig_ref] Table 2: Evaluations of clinical image reconstruction [/fig_ref]. It can be seen that the results show the coherence with the results of numerical phantom. NLTV obtains better RMSE and MSSIM than other methods but suffers from larger computation overheads. # Discussion and conclusion With the development of modern medical imaging technologies, CT has been playing an increasing important role in clinical analysis. Image reconstruction with sparse projection is one of the most efficient ways to lower the dose the patients will endure. CS is a powerful tool to deal with this problem. CS has proved that a complete signal can The Scientific World Journal be recovered, while a sparsifying transform exists. In this situation, Nyquist sampling theory may not be fit. TV is widely used as an efficient sparsifying transform and it can be introduced into many topics in CT reconstruction, such as sparse projection, limited-angle, and interior reconstruction. Although TV is popular, the blocky effect in homogeneous regions limits the applications in clinical practice. The main reason for this phenomenon is that TV is neighborhood based and there is no global information involved. This will lead to loss of structure and texture information. To solve this problem, we introduce NLTV into medical imaging and give its application in sparse-projection reconstruction. NLTV calculates the weights by searching in all the image patches and it avoids being dependent only on neighboring pixels. The experimental results show that the presented NLTV method can yield more significant performance gains than the existing methods, including FBP, EM, and ASD-POCS, in terms of visual effect and different measurement metrics. There are several parameters in our methods. All of them should be determined manually, namely, the search window size , the patch window size , the filtering parameters , and the regularization scale parameter . Note that, all the parameters are application related. In our purpose, a bigger theoretically means that more similarity information will be acquired. By extensive experiments, = 21 × 21 and = 5 × 5 will be appropriate settings for effective noise and artifact suppression while maintaining computational efficiency. For the other parameters, and , in our simulations, we simply select the best average configuration based on the results obtained with a broad range of parameter values manually in terms of visual inspection and quantitative measurements. Due to the computational cost of the proposed method, an adaptive mechanism will be useful and in the future work, we will focus on this problem. [formula] 5 (a) (b) (c) (d) (e) (f) [/formula] Another concern is the computation cost of the proposed method. As a result of introducing global patch distance computation, the computational burden is much bigger than other iteration based methods. However, with the rapid development of storage hardware, the processing time will not be main obstacle and also the proposed method can be implemented on PC clusters or on graphic processing units (GPU), which will make it feasible for practical application. The Scientific World Journal In conclusion, in this paper, we present a novel sparseprojection image reconstruction method using nonlocal total variation. After experiments on numerical phantoms and clinical cases, the proposed method shows better performance than several commonly used methods with respect to both quantitativeness and qualitativeness. Although the computational cost of this method is larger than current methods, there are several methods that can accelerate the processing speed. It will be convenient to implement and add to modern CT systems. The optimization of adaptive parameter selection and acceleration is another concern in our future work. [fig] Figure 1: Fan-beam CT geometry configuration. [/fig] [fig] a initialization of performance parameters and,: for = 1, . . . , ,+, = ∑ =1, , for = 1, . . . , ; [/fig] [fig] Figure 2: (c) is not satisfactory. The whole image is fulfilled with severe artifacts and it is difficult to recognize any parts of the phantom. In Figures 2(d) and 2(e), ASD-POCS and NLTV suppress most of the artifacts. ASD-POCS recovers all the virtual organs except some structural parts. As we marked with white arrows in Figures 2(d) and 2(e), when ASD-POCS dealt with regions full of small structure information, oversmoothing effect appeared. Different parts with very small intervals are obscure and some Reconstruction results of Shepp-Logan phantom by different methods. (a) Original, (b) FBP, (c) EM, (d) ASD-POCS, and (e) NLTV. [/fig] [fig] Figure 3: Reconstruction results of chest image by different methods. (a) Original, (b) FBP, (c) EM, (d) ASD-POCS, (e) NLTV, and (f) zoomed parts of original image, ASD-POCS, and NLTV. First row is original images, the second row is ASD-POCS results, and the third row is NLTV results. [/fig] [table] Table 1: Evaluations of numerical phantom reconstruction. [/table] [table] Table 2: Evaluations of clinical image reconstruction. [/table]

Characterization of Inner Retinal Hyperreflective Alterations in Early Cognitive Impairment on Adaptive Optics Scanning Laser Ophthalmoscopy Citation: Zhang YS, Onishi AC, Zhou N, et al. Characterization of inner retinal hyperreflective alterations in early cognitive impairment on adaptive optics scanning laser ophthalmoscopy. Invest Ophthalmol Vis Sci. 2019;60:3527-3536. https://doi.org/ 10.1167/iovs.19-27135PURPOSE. To examine inner retinal hyperreflective features on adaptive optics scanning laser ophthalmoscopy (AOSLO) in individuals with early cognitive impairment.METHODS.In this prospective, cross-sectional study, we enrolled 12 participants with either amnestic mild cognitive impairment (aMCI, n ¼ 10) or early dementia due to Alzheimer's disease (eAD, n ¼ 2) and 12 age-, sex-, and race-matched cognitively normal controls. All participants completed AOSLO imaging of the inner retina. AOSLO montages of the peripapillary area were graded for hyperreflective features including granular membranes, mottled membranes, and nummular features. Regions of interest on AOSLO were compared qualitatively to corresponding optical coherence tomography (OCT) cross sections. OCT was also used to analyze peripapillary retinal nerve fiber layer (RNFL) thickness.RESULTS.Cognitively impaired individuals had a significantly higher number of granular membranes with a larger overall area compared to controls. The proportion of cognitively impaired individuals with two or more granular membranes was 41.7% compared to none in the control group. Granular membrane area was also inversely correlated with cognitive performance on the Montreal Cognitive Assessment. There was no difference between the two groups in terms of other membrane types or RNFL thickness.CONCLUSIONS. Individuals with early cognitive impairment related to Alzheimer's show hyperreflective granular membranes on high-resolution imaging, which we hypothesize to be manifestations of inner retinal gliosis. The presence of these subtle hyperreflective membranes may obscure underlying RNFL thinning in these eyes on OCT imaging. The distinctive phenotype of granular membranes surrounding the optic nerve on AOSLO may represent a new potential biomarker of early Alzheimer's. A lzheimer's disease (AD) is a neurodegenerative disease with hallmark extracellular amyloid plaques that involve not only the brain but also the inner retina. [bib_ref] Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease, Koronyo [/bib_ref] The inner retina bears the brunt of pathology, confirmed by histopathological studies that identified optic nerve axon degeneration and macular loss of retinal ganglion cells (RGCs) in the eyes of patients with AD. [bib_ref] Retinal pathology in Alzheimer's disease. I. Ganglion cell loss in foveal/ parafoveal..., Blanks [/bib_ref] [bib_ref] Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes..., Blanks [/bib_ref] Before the onset of dementia due to AD, patients may be identified as having mild cognitive impairment (MCI) based on neurocognitive testing. [bib_ref] The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from..., Albert [/bib_ref] Those with amnestic type MCI (amnestic mild cognitive impairment have up to a 48.7% chance of converting to AD within 30 months and thus represent an important target population for early intervention and disease surveillance. [bib_ref] Conversion from subtypes of mild cognitive impairment to Alzheimer dementia, Fischer [/bib_ref] Several studies have examined inner retinal changes on optical coherence tomography (OCT) in individuals with MCI in hopes of finding early biomarkers of the disease. [bib_ref] Optical coherence tomography in Alzheimer's disease: a meta-analysis, Coppola [/bib_ref] [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] [bib_ref] Retinal ganglion cell analysis using high-definition optical coherence tomography in patients with..., Cheung [/bib_ref] [bib_ref] Evaluation of inner retinal layers as biomarkers in mild cognitive impairment to..., Lad [/bib_ref] However, results have been highly variable, showing decreased, [bib_ref] Optical coherence tomography in Alzheimer's disease: a meta-analysis, Coppola [/bib_ref] unremarkable, 9 or increased 7 retinal nerve fiber layer (RNFL) thickness in MCI. Interestingly, in AD individuals with more advanced cognitive impairment, RNFL thinning is a universal finding, suggesting that this may be a late finding in these eyes. [bib_ref] Optical coherence tomography in Alzheimer's disease: a meta-analysis, Coppola [/bib_ref] [bib_ref] Retinal ganglion cell analysis using high-definition optical coherence tomography in patients with..., Cheung [/bib_ref] One potential explanation for the wide variability in RNFL measurements could be related to the occurrence of reactive gliosis in the inner retina in MCI and early stages of AD. [bib_ref] Reactive nonproliferative gliosis predominates in a chronic mouse model of glaucoma, Inman [/bib_ref] We hypothesize that gliosis in these early stages may mask underlying subtle RNFL thinning on OCT since hypertrophy and proliferation of glial cells both within and on the surface of the RNFL may lead to artifactual thickening of the RNFL. [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] [bib_ref] Nerve fiber layer astrocytes of the primate retina: morphology, distribution, and density, Ogden [/bib_ref] Gliosis is a common inflammatory response involving hypertrophy, proliferation, and functional changes in glial cells in response to stress. [bib_ref] Retinal glial changes in Alzheimer's disease -a review, Fernandez-Albarral [/bib_ref] This response has been reported in the brain and eye in chronic neurodegenerative diseases such as Parkinson's disease and AD and in glaucoma. [bib_ref] The role of microglia in retinal neurodegeneration: Alzheimer's disease, Parkinson, and glaucoma, Ramirez [/bib_ref] [bib_ref] Deciphering the astrocyte reaction in Alzheimer's disease, Perez-Nievas [/bib_ref] [bib_ref] Detection of retinal glial cell activation in glaucoma by time domain optical..., Grieshaber [/bib_ref] Histopatho-logical studies of AD have reported reactive astrocytes clustered around amyloid-b (Ab) plaques in the brain [bib_ref] Deciphering the astrocyte reaction in Alzheimer's disease, Perez-Nievas [/bib_ref] and increased glial cell number and reactivity in the retina. [bib_ref] Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes..., Blanks [/bib_ref] [bib_ref] Retinal glial changes in Alzheimer's disease -a review, Fernandez-Albarral [/bib_ref] In primary open-angle glaucoma, presumed activated retinal astrocytes and Müller cells appear on OCT as patchy hyperreflective structures on the RNFL that may mask RNFL thinning. [bib_ref] Detection of retinal glial cell activation in glaucoma by time domain optical..., Grieshaber [/bib_ref] [bib_ref] Retinal putative glial alterations: implication for glaucoma care, Ashimatey [/bib_ref] Hyperreflective retinal alterations that may suggest gliosis have not been studied in vivo in the eyes of those with ADrelated dementia or in MCI. Recent advancements in adaptive optics scanning laser ophthalmoscopy (AOSLO) allow noninvasive visualization of the RNFL in vivo. [bib_ref] High-resolution imaging of the retinal nerve fiber layer in normal eyes using..., Takayama [/bib_ref] This technique reveals inner retinal features at resolutions that are not possible with OCT. [bib_ref] High-resolution imaging of the retinal nerve fiber layer in normal eyes using..., Takayama [/bib_ref] [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] Scoles et al. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] have used AOSLO to characterize seven categories of hyperreflective inner retinal structures in participants with neurological diseases other than AD. In this prospective case-control study, we used AOSLO to investigate hyperreflective signatures in a population with early cognitive impairment (aMCI or early dementia due to AD (eAD) and compared them to cognitively normal, matched controls. # Methods ## Study population This single-center case-control study at the Department of Ophthalmology, Northwestern University in Chicago, Illinois, was prospectively approved by the Institutional Review Board of Northwestern University. The study was conducted in accordance with the tenets of the Declaration of Helsinki. We used the University of California, San Diego Brief Assessment of Capacity to Consent (UBACC) to assess capacity to provide informed consent and obtained written informed consent from all participants. We recruited a total of 16 cognitively impaired individuals with a clinical diagnosis of aMCI 4 (n ¼ 14 participants) or eAD [bib_ref] The diagnosis of dementia due to Alzheimer's disease: recommendations from the National..., Mckhann [/bib_ref] (n ¼ 2 participants) and 14 matched cognitively normal controls from the Clinical Core of the Northwestern Alzheimer's Disease Center (ADC). We combined aMCI and eAD participants in this cohort because of the presumed shared underlying cause of their cognitive impairment and similar severity of impairment (mild). [bib_ref] The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from..., Albert [/bib_ref] We specifically focused on aMCI participants, defined as MCI individuals with a predominant memory domain deficit. The clinical diagnoses of aMCI and eAD were based on previously published diagnostic criteria from the National Institute on Aging. [bib_ref] The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from..., Albert [/bib_ref] [bib_ref] The diagnosis of dementia due to Alzheimer's disease: recommendations from the National..., Mckhann [/bib_ref] Briefly, all aMCI and eAD diagnoses required a detailed research clinical assessment to rule out additional medical causes of impairment and a formal neuropsychological battery from the Uniform Data Set (UDS) as described in the Cognitive Assessment section. Whenever possible, available biomarker data were also included in consideration of the final diagnosis. [bib_ref] The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from..., Albert [/bib_ref] [bib_ref] The diagnosis of dementia due to Alzheimer's disease: recommendations from the National..., Mckhann [/bib_ref] The ADC provided available cerebral spinal fluid (CSF) biomarker status, determined based on Ab 1-42 and phosphorylated-tau levels as previously described, [bib_ref] Cerebrospinal fluid markers detect Alzheimer's disease in nonamnestic dementia, Oboudiyat [/bib_ref] and apolipoprotein E e4 (apoE4) allele status, a genetic risk factor of AD. [bib_ref] Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy, Liu [/bib_ref] [bib_ref] Results from the NACC Uniform Data Set Neuropsychological Battery Crosswalk Study, Monsell [/bib_ref] to be categorized as early in their disease course. In addition to specific neurocognitive performance metrics, the diagnosis of MCI required preservation of activities of daily living while eAD required an impairment of activities of daily living due to the cognitive loss. Thus, all participants had a study partner who completed the Activities of Daily Living Questionnaire, a reliable assessment of functional ability in dementia. [bib_ref] An examination of instrumental activities of daily living assessment in older adults..., Gold [/bib_ref] [bib_ref] The Activities of Daily Living Questionnaire: a validation study in patients with..., Johnson [/bib_ref] Cognitively normal controls also underwent the same battery of assessments. Control individuals were matched to the cognitively impaired individuals based on age (within 3 years), sex, and race. Exclusion criteria included pre-existing ocular pathology such as glaucoma, macular degeneration, diabetic or hypertensive retinopathy, retinal detachment, ocular trauma, high myopia (>6 diopters [D]), intraocular pressure (IOP) of >20, epiretinal membranes or extensive cataracts confirmed by participant report, OCT images, and review of electronic medical records. Participants meeting any of these exclusion criteria by personal report, OCT images, or on review of medical records were excluded. We also excluded participants with neurological disorders known to affect the retina such as multiple sclerosis and Parkinson's disease, as well as conditions such as uncontrolled hypertension or diabetes, and current smokers. On AOSLO, we excluded eyes that had less than one quadrant imaged or poor-quality images with the RNFL not in focus. On OCT, we excluded images with a signal quality measure of <6 or an absolute signal strength intensity <45. ## Cognitive assessment All participants had completed the same extensive annual neurocognitive assessment using the Uniform Data Set (UDS) of the National Institute on Aging Alzheimer's Disease Program. [bib_ref] Version 3 of the Alzheimer Disease Centers' Neuropsychological Test Battery in the..., Weintraub [/bib_ref] Testing included a battery of clinical, functional, and neuropsychological assessments in the domains of language, visuospatial functions, executive attention, and working memory. [bib_ref] Version 3 of the Alzheimer Disease Centers' Neuropsychological Test Battery in the..., Weintraub [/bib_ref] In the current study, we focused on memory domain tests including the Craft Story 21 immediate and delayed recall, which assess episodic memory, and the Rey Audio Verbal Learning Test delayed recall, which assesses word list memory. The MoCA is also included as a measure of overall cognitive function. We also report the global score on the Clinical Dementia Rating (CDR) scale, a clinical metric of cognitive-functional impairment. Amnestic MCI is usually defined by a global CDR score of 0.5 and eAD by a score of 0.5 to 1.0 (mild dementia). [bib_ref] The Clinical Dementia Rating (CDR): current version and scoring rules, Morris [/bib_ref] On all reported neuropsychological tests, a higher score indicates better performance. ## Ophthalmologic assessment We obtained a full history and reviewed the electronic health record of all participants for past medical, ocular, surgical, and medication (including eye drops) history that may suggest a confounding ocular disease such as glaucoma. All participants underwent manual refraction, best-corrected visual acuity, confrontational visual field testing, and IOP measurement using Tono-Pen (Reichert Technologies, Buffalo, NY, USA). All imaging was performed after pupil dilation (proparacaine 0.5%, tropicamide 1%, and phenylephrine 2.5% ophthalmic solutions). Macular and optic nerve pathology was assessed on OCT scans by a board-certified ophthalmologist, retina specialist (A.A.F.). ## Adaptive optics imaging We acquired AOSLO images using the Apaeros LF retinal imaging system (Boston Micromachines Corporation, Boston, MA, USA) based on the optical design of Dubra et al. [bib_ref] Reflective afocal broadband adaptive optics scanning ophthalmoscope, Dubra [/bib_ref] As previously described, [bib_ref] Characterization and correlation of ''Jampol dots'' on adaptive optics with foveal granularity..., Onishi [/bib_ref] the system uses an 97 actuator AlpAO DM (AlpAO SAS, Montbonnot, France) with 25 lm of stroke for wavefront correction. The light sources include two superluminescent diodes, centered at 790 and 850 nm. The 790-nm source was used in imaging and the 850-nm source for wavefront sensing. The combined power at the eye was~130 lW. Our AOSLO images were focused on the inner retina, specifically the inner surface of the RNFL, which was identified by the characteristic reflective retinal nerve bundle striations and vasculature as previously shown. [bib_ref] High-resolution imaging of the retinal nerve fiber layer in normal eyes using..., Takayama [/bib_ref] [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] We began image acquisition at the optic nerve head and continued in a grid pattern, guided by a widefield mirror to capture the superior, temporal, and inferior peripapillary regions. We started with widefield 58 3 4.58 image sequences of the RNFL layer for orientation, then acquired 28 3 28 scans. In areas of interest, we also obtained additional high-resolution 18 3 18 images. Images were acquired in sequences of 60 frames over 2 seconds. To facilitate montaging, we used an overlap of 0.58 between image sequences. ## Optical coherence tomography imaging We acquired structural RNFL thickness using the RTVue-XR OCT Avanti System (Optovue, Inc., Fremont, CA, USA. Software Version 2016.1.0.26). We also examined the peripapillary 4.5 3 4.5-mm 2 en face OCT and cross-sectional B-scans qualitatively for inner retinal pathology corresponding to regions of interest on AOSLO. ## Adaptive optics image analysis We uploaded the AOSLO images onto ImageJ 29 and extracted representative frames (individual images from the image sequence) of each region of the RNFL for semiautomated montage of the individual frames using the i2k Retina Pro montaging software (DualAlign LLC, Clifton Park, NY, USA). Two separate graders (Y.S.Z., A.C.O.), masked to the diagnosis of the participants, identified hyperreflective features on the montages. They categorized the features based on their size, location, and phenotypic appearance and measured their area and dimensions. In addition, we measured the total retinal area imaged in each participant. All measurements were performed on Image J and calculated based on a theoretical (not actual) average axial length of 24 mm. ## Membrane types Membranes with previously reported characteristics were categorized based on specifications by Scoles et al. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] Granular membranes were defined as a contiguous hyperreflective structure >50 lm in diameter with texture similar to clustered grains of sand, forming a mesh-like membrane with distinct borders. Nummular features were defined as a discrete discshaped hyperreflectivity 10 to 30 lm in diameter. Membranes that did not fit into previously reported types were named by agreement. For example, we identified mottled membranes that did not fit into either the waxy-noted as a very smooth and opaque membrane-or granular membranes described by Scoles et al. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] We defined mottled membranes as >50 lm in diameter and obscuring the underlying RNFL with some areas of clearing. Their texture was coarser compared to waxy or granular membranes, nonhomogeneous, and speckled with scattered punctate areas of reflectivity >10 lm in diameter. # Statistical analysis All statistical analyses were performed using SPSS24 (IBM Corp., Armonk, NY, USA). An interclass correlation (ICC) for absolute agreement was performed for the image analysis results completed by the two separate graders. A P value of <0.05 was considered significant for all analyses. We used the Mann-Whitney U test for nonparametric comparison of the number and area covered by hyperreflective membranes between the cognitively normal and impaired groups as well as between the CSF biomarker or apoE4 allele present and absent group. We used Fisher's exact test for small sample sizes to compare the proportion of individuals in each group (cognitively impaired or control) with membranes. Spearman correlation for nonparametric populations was used to investigate the correlation between membrane parameters and cognitive performance or age. We did not compare nummular features between our cohorts as these features are prevalent in both healthy and diseased states. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] # Results Our study originally recruited 30 participants (aMCI n ¼ 14, eAD n ¼ 2, control n ¼ 14 participants). One aMCI individual was excluded for inability to follow the target to complete imaging. Two aMCI and two control participants were excluded for bilateral poor image quality, likely due to extensive cataracts. Finally, one aMCI individual was excluded for findings of intraretinal edema on OCT. Ultimately, 10 aMCI and 2 eAD participants were eligible for further analysis. Six individual eyes (5 eyes due to poor image quality, 1 eye for fixation difficulties) were excluded, leaving 18 eyes in the cognitively impaired group for final analysis. In the 12 control participants, 3 eyes were excluded due to poor image quality, leaving 21 eyes as shown on [fig_ref] TABLE 1: Demographics and Cognitive Information [/fig_ref]. Overall, the number of eyes and the peripapillary area successfully imaged were not significantly different between the two groups. Of note, only 5/12 of the cognitively impaired and 10/12 of the control participants had complete imaging of the superior quadrant of the optic nerve due to machine limitations. Other demographic information shows that the two groups were successfully matched according to age, sex, and race and had similar IOP and interval between cognitive testing and imaging visits. As expected, the cognitively impaired group performed worse on all cognitive tests. The two graders showed excellent absolute ICC with coefficient of 0.930 (95% confidence interval [CI] at 0.831-0.971). A total of 26 membranes were found in our 24 participants (39 total eyes). Most of the membranes (20/26) were found in the inferior quadrant while 2 granular membranes were found in the temporal quadrant and 4 granular membranes were found in the superior quadrant of eyes with aMCI. [fig_ref] TABLE 2: Comparison of RNFL Thickness and Membrane Parameters Between the Cognitively Impaired and... [/fig_ref] summarizes the overall results for hyperreflective membranes and RNFL thickness. [fig_ref] TABLE 3: Spearman Correlation Between Membrane Parameters, Cognitive Performance, and Age [/fig_ref] summarizes the correlation between membrane parameters and participant age or cognitive score. Available apoE4 data were obtained from 21 participants. Seven of the 12 (58.3%) cognitively impaired individuals had at least 1 apoE4 allele while none of the 9 controls with available genetic data had any apoE4 alleles. Five of our cognitively impaired participants had CSF biomarker data available and were all biomarker positive. When considering the genetic and biomarker data together, nine (75%) cognitively impaired participants had at least one apoE4 allele or CSF biomarker present. Thirteen of the remaining participants had no genetic allele or CSF biomarkers. [fig_ref] TABLE 4: Comparison of Membrane Parameters Between the apoE4 or CSF Present and Absent... [/fig_ref] summarizes our findings comparing membrane parameters between the cohort with either genetic allele or CSF biomarker present and the participants with both parameters absent. [fig_ref] FIGURE 1: Granular membranes on adaptive optics scanning laser ophthalmoscopy [/fig_ref] shows representative images of granular membranes. On AOSLO, the granular membranes showed a sand-like texture and did not follow the direction of the RNFL. The membranes covered the RNFL, most often adjacent to the vasculature. On [fig_ref] FIGURE 2: Granular membranes on multimodal imaging [/fig_ref] , corresponding areas on OCT cross sections (Figs. 2G-I) showed that a minority of granular membranes (4/21 membranes, 19.0%) could be visualized on OCT as preretinal hyperreflective layers, while the majority of membranes were not discernable on OCT. Four of the aMCI participants with granular membranes also had noncontiguous, poorly defined satellite hyperreflective lesions surrounding the primary well-demarcated granular membrane, as shown in [fig_ref] FIGURE 1: Granular membranes on adaptive optics scanning laser ophthalmoscopy [/fig_ref] in blue arrows. ## Granular membranes We found that in the group with cognitive impairment, 7/12 (58.3%) participants had granular membranes (19 membranes total) while only 2/12 (16.7%) control participants had granular membranes (2 membranes total), as summarized in [fig_ref] TABLE 2: Comparison of RNFL Thickness and Membrane Parameters Between the Cognitively Impaired and... [/fig_ref]. We found that a statistically significant (P ¼ 0.037) proportion of cognitively impaired individuals had more than one granular membrane (2þ membranes) as compared to the control group, with a large effect size of Cramer's V ¼ 0.513. However, there was no difference in the number of participants in either group with one membrane or more (1þ membrane). We also found that the cognitively impaired group had more membranes (P ¼ 0.020; Hedges' g effect size ¼ 0.902) and wider area covered by granular membranes (P ¼ 0.020; Hedges' g effect size ¼ 0.879) compared to the controls. [fig_ref] TABLE 3: Spearman Correlation Between Membrane Parameters, Cognitive Performance, and Age [/fig_ref] summarizes a statistically significant inverse correlation (r ¼À0.411, P ¼ 0.046) between the overall extent of granular membranes and cognitive performance in the entire cohort. We found that individuals with a larger area of granular membranes had worse performance on the MoCA. Lastly, while [fig_ref] TABLE 4: Comparison of Membrane Parameters Between the apoE4 or CSF Present and Absent... [/fig_ref] shows no statistically significant differences between participants with genetic or CSF biomarkers and those without, the cohort with apoE4 or CSF biomarker present had a higher mean number (P ¼ 0.051) and area (P ¼ 0.060) of granular membranes that was approaching significance. ## Mottled membranes Figures 3A and 3B show mottled membranes, which were found in the cognitively impaired (n ¼ 2 participants) and control (n ¼ 1 participant) groups. Mottled membranes showed a coarse and lacy appearance with scattered and larger punctate areas of hyperreflectivity, often appearing nonhomogeneous, which distinguished them from granular membranes. All of the mottled membranes were found in the inferior quadrant. There were no corresponding alterations visualized on OCT. We did not find a statistically significant difference in these membranes between the cognitively normal and impaired groups or between the CSF biomarker or apoE4 allele present and absent groups. ## Nummular features We found clusters of nummular hyperreflective features (Figs. 3C, 3D) in both aMCI (n ¼ 4 participants) and control (n ¼ 4 participants) individuals. These disc-shaped granular textured reflective features appeared on the inner retina with a diameter of 10 to 30 lm and often had a central clearing, as previously described. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] # Discussion The major finding of our study is that individuals with early cognitive impairment related to suspected AD have more numerous and larger areas covered by granular membranes, which we hypothesize are manifestations of inner retinal gliosis on AOSLO. These membranes (up to 500 lm in diameter) are much larger than the solid ovoid retinal amyloid plaques (1-10 lm) that have previously been reported in AD histopathological studies. [bib_ref] Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease, Koronyo [/bib_ref] [bib_ref] Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in..., Koronyo-Hamaoui [/bib_ref] We hypothesize that these preretinal granular membranes [fig_ref] FIGURE 1: Granular membranes on adaptive optics scanning laser ophthalmoscopy [/fig_ref] could be a manifestation of reactive gliosis, related either to Müller cell end feet that normally form the internal limiting membrane, activated glial cells that have migrated to a location of stress, [bib_ref] Müller cell activation, proliferation and migration following laser injury, Tackenberg [/bib_ref] or reactive astrocytic processes that normally surround blood vessels in the inner retina. Previous studies have established an association between AD and glial cell activation in the brain and retina. [bib_ref] Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes..., Blanks [/bib_ref] [bib_ref] The role of microglia in retinal neurodegeneration: Alzheimer's disease, Parkinson, and glaucoma, Ramirez [/bib_ref] In the human AD retina, Blanks et al. [bib_ref] Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes..., Blanks [/bib_ref] described extensive labeling of Müller cell radial processes and end feet in addition to proliferation and activation of astrocytes. Furthermore, an animal model of AD has shown histologically dense bundles of Müller cell end feet forming a network surrounding the blood vessels of the inner retina. [bib_ref] Retinal macroglia changes in a triple transgenic mouse model of Alzheimer's disease, Edwards [/bib_ref] In the MCI population, our group previously found an inverse correlation between RNFL thickness in the inferior quadrant and cognitive performance, leading us to hypothesize that reactive gliosis may be an earlier feature than previously observed. [bib_ref] Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes..., Blanks [/bib_ref] [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] Many studies have explored whether RNFL measurements could serve as an early marker for detection of AD. [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] [bib_ref] Evaluation of inner retinal layers as biomarkers in mild cognitive impairment to..., Lad [/bib_ref] [bib_ref] A systematic review and meta-analysis of retinal nerve fiber layer change in..., Thomson [/bib_ref] [bib_ref] Analysis of the retinal nerve fiber layer thickness in Alzheimer disease and..., Kwon [/bib_ref] However, the results of these studies have been highly controversial, showing either thinner, unremarkable, or thicker RNFL measurements in MCI participants compared to controls. [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] [bib_ref] Evaluation of inner retinal layers as biomarkers in mild cognitive impairment to..., Lad [/bib_ref] [bib_ref] A systematic review and meta-analysis of retinal nerve fiber layer change in..., Thomson [/bib_ref] [bib_ref] Analysis of the retinal nerve fiber layer thickness in Alzheimer disease and..., Kwon [/bib_ref] In the current cohort, we found no difference in RNFL thickness between the cognitively impaired and control groups [fig_ref] TABLE 2: Comparison of RNFL Thickness and Membrane Parameters Between the Cognitively Impaired and... [/fig_ref]. This finding corroborates our prior study [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] and further supports the hypothesis that reactive gliotic changes in early cognitive impairment may mask or precede pathological neurodegeneration that is ultimately detectable in later stages of AD as RNFL thinning. [bib_ref] Optical coherence tomography in Alzheimer's disease: a meta-analysis, Coppola [/bib_ref] [bib_ref] A systematic review and meta-analysis of retinal nerve fiber layer change in..., Thomson [/bib_ref] If the initial change in the retina of cognitively impaired individuals is indeed reactive gliosis, then this may explain the highly variable reports of RNFL changes in MCI groups [bib_ref] Optical coherence tomography in Alzheimer's disease: a meta-analysis, Coppola [/bib_ref] [bib_ref] Retinal nerve fiber layer thickness in amnestic mild cognitive impairment: case-control study..., Knoll [/bib_ref] [bib_ref] Evaluation of inner retinal layers as biomarkers in mild cognitive impairment to..., Lad [/bib_ref] and would call for a paradigm shift in the search for retinal biomarkers that could be ideal for early detection. Our current study highlights granular membranes, a potential manifestation of gliosis on AOSLO, as a candidate biomarker for early cognitive impairment. Another avenue for biomarkers is the retinal vasculature, which can be visualized noninvasively by OCT angiography (OCTA). [bib_ref] Optical coherence tomography angiography: a comprehensive review of current methods and clinical..., Kashani [/bib_ref] Several groups have found decreased parafoveal vessel density and flow on OCTA in those with more advanced AD, especially in rigorous studies that accounted for potential age-related changes. [bib_ref] Altered macular microvasculature in mild cognitive impairment and Alzheimer disease, Jiang [/bib_ref] [bib_ref] Evaluation of optical coherence tomography angiographic findings in Alzheimer's type dementia, Bulut [/bib_ref] According to the vascular hypothesis of AD, early hypoperfusion in AD may lead to decreased Ab clearance and subsequent plaque accumulation, [bib_ref] The nature and effects of cortical microvascular pathology in aging and Alzheimer's..., Bailey [/bib_ref] [bib_ref] Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease, Kisler [/bib_ref] and thus, vascular alterations could be early manifestations in the course of the disease. Indeed, our group recently showed that individuals with early cognitive impairment also exhibit inner retinal hypoperfusion that manifests on OCTA as decreased parafoveal vessel density. [bib_ref] Parafoveal vessel loss and correlation between peripapillary vessel density and cognitive performance..., Zhang [/bib_ref] Our current finding of granular membranes on AOSLO and previous study of vascular hypoperfusion on OCTA provide promising biomarkers of early AD that warrant large-scale investigations. A secondary finding in our study is an association between granular membrane area and poor cognitive performance [fig_ref] TABLE 3: Spearman Correlation Between Membrane Parameters, Cognitive Performance, and Age [/fig_ref] , which further suggests that these membranes may serve as biomarkers. However, longitudinal studies are needed to further investigate the relationship between these membranes and conversion to AD or disease progression. It is possible that the satellite lesions seen in a few aMCI and eAD participants are secondary extensions of gliosis that may eventually become contiguous components of the primary granular membrane, a question to be explored in longitudinal studies. We also found a trend toward significantly increased number and area of granular membranes in participants with either the apoE4 allele or CSF biomarkers of AD, a trend that did not exist for mottled membranes. ApoE is a cholesterol carrier that has been implicated in Ab metabolism. The apoE4 allele is one of the strongest genetic risk factors for AD, with the odds ratio of developing disease reported to range from 14.9 to 33.1 in individuals with two alleles. [bib_ref] Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy, Liu [/bib_ref] [bib_ref] Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's..., Lambert [/bib_ref] [bib_ref] Effects of age, sex, and ethnicity on the association between apolipoprotein E..., Farrer [/bib_ref] CSF biomarker positivity provides indirect evidence of AD pathology in MCI and AD patients, with a positive predictive value for conversion to AD of 60% to 70% in individuals with MCI. [bib_ref] CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment, Mattsson [/bib_ref] [bib_ref] Evaluation of CSF biomarkers as predictors of Alzheimer's disease: a clinical follow-up..., Hertze [/bib_ref] Therefore, individuals with either the apoE4 allele or CSF biomarkers represent a high-risk population for having or developing AD pathology. The trend of increased granular but not mottled membrane parameters in these high-risk individuals further supports the potential role of granular membrane in bolstering current biomarker and genetic data to risk stratify patients. Future studies correlating AOSLO findings to complete Alzheimer's biomarker data should be considered. Beyond our findings in cognitively impaired individuals, our study reflects on the relevance of exploring the inner retina using AOSLO. Previously, hyperreflective alterations superficial to the RNFL on OCT have been putatively linked to activated glial cells in diseases such as glaucoma [bib_ref] Retinal putative glial alterations: implication for glaucoma care, Ashimatey [/bib_ref] [bib_ref] Gliosis-like retinal alterations in glaucoma patients, Graf [/bib_ref] and retinitis pigmentosa, [bib_ref] Spectral-domain optical coherence tomography reveals prelaminar membranes in optic nerve head pallor..., Rashaed [/bib_ref] and in normal aging. [bib_ref] Retinal putative glial alterations: implication for glaucoma care, Ashimatey [/bib_ref] [bib_ref] Epiretinal gliosis, Haritoglou [/bib_ref] AOSLO has a much higher lateral resolution than OCT, allowing for more specific characterization of the surface features of these hyperreflective structures, [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] which is critical in illustrating their pathological signatures. For example, on AOSLO epiretinal membranes appear striated and contracted, [bib_ref] Adaptive optics imaging of idiopathic epiretinal membranes, Lombardo [/bib_ref] while waxy membranes appear smooth like dripped wax. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] Both of these membranes are phenotypically distinct from the granular or mottled membranes described in our study, a fine distinction that far exceeds the resolution possible on OCT. In fact, the majority of our membranes .6%) showed no corresponding alterations on cross-sectional OCT. Thus, our study highlights the importance of using AOSLO, to better define the phenotypic and pathological spectrum of inner retinal hyperreflective signatures. On AOSLO, Scoles et al. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] previously showed that granular membranes can be seen in several disease states including glaucoma and Parkinson's, where histopathological evidence of gliosis in the retina has been reported. [bib_ref] The role of microglia in retinal neurodegeneration: Alzheimer's disease, Parkinson, and glaucoma, Ramirez [/bib_ref] [bib_ref] Early microglia activation in a mouse model of chronic glaucoma, Bosco [/bib_ref] Therefore, we believe that granular membranes could be a manifestation of a common gliotic pathway shared by several disease entities. Our data [fig_ref] TABLE 2: Comparison of RNFL Thickness and Membrane Parameters Between the Cognitively Impaired and... [/fig_ref] showed that a greater proportion of cognitively impaired participants had multiple granular membranes (n ¼ 5 participants, 41.7%) compared to controls (n ¼ 0 participants). Although a small minority of controls (n ¼ 2 participants, 16.7%) also had granular membranes, they each had a single small membrane. The presence of membranes in our control group is not surprising when considering the many pathways that lead to gliotic changes. In fact, hyperreflective alterations can be found on OCT in healthy eyes, especially with advancing age. [bib_ref] Retinal putative glial alterations: implication for glaucoma care, Ashimatey [/bib_ref] [bib_ref] Gliosis-like retinal alterations in glaucoma patients, Graf [/bib_ref] [bib_ref] Epiretinal gliosis, Haritoglou [/bib_ref] Perhaps taking into account the number and extent of membranes rather than the presence of a single small membrane may be an important consideration in future studies exploring granular membranes as biomarkers. In our study, we intentionally excluded patients with ocular pathology such as retinal detachment and glaucoma that may be associated with gliosis. We also found no significant correlation between age and membrane parameters, suggesting that the hyperreflective alterations seen in our cognitively impaired group cannot be explained by aging. The granular membranes were mostly found near vessels around the optic disc and not associated with any contraction, distinguishing them from epiretinal membranes that have a distinctive striated and contracted appearance on AOSLO. [bib_ref] Adaptive optics imaging of idiopathic epiretinal membranes, Lombardo [/bib_ref] [bib_ref] Relationship between retinal glial cell activation in glaucoma and vascular dysregulation, Grieshaber [/bib_ref] However, based on our data, we were not able to determine the status of posterior vitreous adherence in our participants. Thus, we were unable to comment on the possible contribution of the vitreous to our findings, which may be interesting to consider for future studies. Other hyperreflective alterations found in both groups include nummular reflective features that have previously been found in both healthy and pathological retinas. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] They were hypothesized to be Gunn's dots or physiological Müller glial cell end feet. [bib_ref] Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease, Scoles [/bib_ref] We also found mottled membranes with no difference (number or size) between the two groups. Our data suggest that the distinction between granular and mottled membranes, made possible by AOSLO, appears to be important in the study of AD. Our study paves the way for future AOSLO investigations of inner retinal hyperreflective alterations as potential new biomarkers of early AD. Our findings also serve as a window into early disease pathophysiology involving potential gliosis. As the number of individuals living with AD is projected to grow from 55 million to 88 million by 2050, it is critically important to better understand early disease pathophysiology in order to identify biomarkers that are noninvasive and predictive of disease progression.Current CSF and positron emission tomography markers are invasive and costly, and lack the sensitivity and specificity for early disease detection. [bib_ref] Exploring biomarkers for Alzheimer's disease, Sharma [/bib_ref] [bib_ref] Cost-utility of using Alzheimer's disease biomarkers in cerebrospinal fluid to predict progression..., Handels [/bib_ref] [bib_ref] Biomarkers for the early detection and progression of Alzheimer's disease, Counts [/bib_ref] On the other hand, AOSLO is noninvasive and has the ability to capture inner retinal pathology at unparalleled high resolutions in vivo. [bib_ref] High-resolution imaging of the retinal nerve fiber layer in normal eyes using..., Takayama [/bib_ref] Future studies with a larger sample size and longitudinal design will be essential to validate AOSLO as a tool for screening, surveillance, or risk stratification of patients with early cognitive impairment. The results of these studies could help identify a subgroup within the mildly cognitively impaired population that is at higher risk for progression to AD, 4 who could be ideal candidates for early therapeutic intervention. [bib_ref] Exploring biomarkers for Alzheimer's disease, Sharma [/bib_ref] The strengths of our study include the prospective matched (age, sex, and race) control design. In addition, our control group underwent an equally rigorous neurocognitive assessment as the cognitively impaired group. The rigor and reproducibility of our AOSLO data analysis was ensured by two independent masked graders, and the excellent ICC. The biggest limitation of our study is our small sample size, which can be attributed to the stringent patient selection criteria as well as strict image quality requirements. As the average age of our study population was over 70, the presence of cataracts or inability of patients to fixate on the target for the duration of imaging affected AOSLO image quality, which limited complete collection of AOSLO data in all participants. However, the large effect size of our findings, despite the small sample size, suggests that there is a meaningful difference between the cognitively impaired and control groups. Other limitations include technical limitations that precluded superior quadrant image acquisition in all our participants. Since the superior quadrant is thought to be most affected by AD pathology, [bib_ref] Optic-nerve degeneration in Alzheimer's disease, Hinton [/bib_ref] data from this quadrant may have further strengthened our results. Furthermore, technical limitations in superior quadrant imaging stand as a temporary and technically surmountable limitation in the current system that should not be construed as a generalized limitation of the AOSLO approach in future studies. We also did not obtain axial length for correction of magnification error. However, given the relatively large size of membranes and the exclusion of high myopes, we would not expect our results to change significantly with axial length corrections. Lastly, we were unable to obtain biomarker data from all of our participants. In conclusion, we found significantly more granular membranes, hypothesized to be manifestations of gliosis, in the early cognitively impaired group than in their cognitively normal counterparts. Moreover, increasing granular membrane area correlated with worsening cognitive performance. Over-all, our study suggests that individuals with early cognitive impairment related to AD have granular membranes on AOSLO, not detectable on OCT, that may represent a potentially early noninvasive marker of AD. Future directions include studies with larger sample sizes as well as longitudinal studies to examine the ability of these retinal hyperreflective changes to predict future cognitive decline and conversion to AD. [fig] FIGURE 1: Granular membranes on adaptive optics scanning laser ophthalmoscopy (AOSLO). Scale bars: 100 lm. (A-F) AOSLO en face images of granular membranes from cognitively impaired participants; (G, H) granular membranes from controls. Yellow arrows help delineate the primary granular membrane. In (B, E), the blue arrows delineate the satellite lesions. [/fig] [fig] FIGURE 2: Granular membranes on multimodal imaging. Scale bars: 100 lm. (A-C) Adaptive optics scanning laser ophthalmoscopy (AOSLO) en face images of granular membranes. (D-F) The corresponding location of the AOSLO membranes highlighted in white boxes on en face optical coherence tomography (OCT). The OCT images show masked optic nerve heads as automated by the machine. (G-I) The OCT B-scans in the corresponding region of interest. The cross sections in (G, H) with their insets illustrate subtle hyperreflective features above the retina (yellow arrow), while in (I) there is a suggestion of focal thickening of the internal limiting membrane (yellow arrow). [/fig] [fig] FIGURE 3: Mottled membranes and nummular features on adaptive optics scanning laser ophthalmoscopy (AOSLO). Scale bars: 100 lm. (A, B) Representative AOSLO en face images of mottled membranes from controls. (C, D) AOSLO en face nummular features from cognitively impaired individuals. [/fig] [table] TABLE 1: Demographics and Cognitive Information [/table] [table] TABLE 2: Comparison of RNFL Thickness and Membrane Parameters Between the Cognitively Impaired and Control Groups [/table] [table] TABLE 3: Spearman Correlation Between Membrane Parameters, Cognitive Performance, and Age [/table] [table] TABLE 4: Comparison of Membrane Parameters Between the apoE4 or CSF Present and Absent Groups [/table]

N1-methyl-pseudouridine is incorporated with higher fidelity than pseudouridine in synthetic RNAs In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality.To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs-such as pseudouridine (Ψ) and N 1 -methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro.Synthetic messenger RNAs (mRNAs) are a novel modality for vaccines, and they are also being evaluated as a vector for therapeutics 1-3 . Despite there being several advantages over conventional protein-based approaches, mRNA-based therapeutics are still in early stages of development. Instability of the synthetic mRNAs and the immune responses generated against these synthetic molecules have been key hurdles in the adaptation of this technology, particularly for therapeutic applications where prolonged expression from the synthetic molecule is desirable and repeated dosing of the drug product is required 1,2 . The use of chemically modified bases in synthetic mRNAs is an innovation that has allowed for both an ameliorated immune response to the synthetic molecules and increased protein expression from the mRNA, thereby providing an unprecedented opportunity to use synthetic mRNAs as a novel class of therapeutics for a wide range of indications, including two approved vaccines against COVID-19 4,5 . It has been shown that the incorporation of pseudouridine (ψ), N 1 -methyl-pseudouridine (m1ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A) and 2-thiouridine (s2U) into synthetic mRNAs results in reduced immune responses and increased protein expression in vivo 6-14 . Pseudouridine-modified mRNAs have been shown to result in reduced activation of 2′-5′-oligoadenylate synthetase (OAS), RNA-dependent protein kinase (PKR), and toll-like receptors 6,7,10 . Investigation of ψ derivatives with improved pharmacological properties led to the identification of m1ψ, the current benchmark for synthetic mRNA-based applications. m1ψ is a naturally occurring modification found in 18S rRNA and tRNAs 15-17 , and similar to ψ-modified synthetic mRNAs, the presence of m1ψ in synthetic mRNAs has been demonstrated to result in reduced activation of RNA sensors in cells8,12,14,18. Furthermore, the presence of m1ψ in synthetic mRNAs show increased translation efficiency in cell-free extracts, multiple mammalian cell lines, and mouse models8,9,[12][13][14][18][19][20]. The exact mechanism of how m1ψ enhances translation is not well understood but it has been demonstrated that presence of m1ψ, alone or in combination with other chemical modifications, can alter ribosome transit time on the modified mRNA, and can increase the mRNA half-life by altering the secondary structure of the synthetic mRNA7,14,21,22.Current methods to incorporate modified nucleotides in synthetic mRNAs include complete substitution of the standard nucleotide with a chemically modified nucleotide during the process of in vitro transcription (IVT) by single-subunit DNA-dependent RNA polymerases (ssRNAPs; such as T7, T3, and SP6 RNAP) 1 . In contrast to endogenous mRNAs, in which modified nucleotides occur in specific positions in the mRNA 23,24 , in synthetic OPEN N 1 -methyl-pseudouridine is incorporated with higher fidelity than pseudouridine in synthetic RNAs Tien-Hao Chen, Vladimir Potapov, Nan Dai, Jennifer L. Ong & Bijoyita Roy * ## In vitro transcribed synthetic messenger rnas (mrnas) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs-such as pseudouridine (Ψ) and N 1 -methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro. Synthetic messenger RNAs (mRNAs) are a novel modality for vaccines, and they are also being evaluated as a vector for therapeutics [bib_ref] mRNA-based therapeutics-Developing a new class of drugs, Sahin [/bib_ref] [bib_ref] mRNA vaccines-A new era in vaccinology, Pardi [/bib_ref] [bib_ref] mRNA vaccines for infectious diseases: Principles, delivery and clinical translation, Chaudhary [/bib_ref]. Despite there being several advantages over conventional protein-based approaches, mRNA-based therapeutics are still in early stages of development. Instability of the synthetic mRNAs and the immune responses generated against these synthetic molecules have been key hurdles in the adaptation of this technology, particularly for therapeutic applications where prolonged expression from the synthetic molecule is desirable and repeated dosing of the drug product is required [bib_ref] mRNA-based therapeutics-Developing a new class of drugs, Sahin [/bib_ref] [bib_ref] mRNA vaccines-A new era in vaccinology, Pardi [/bib_ref]. The use of chemically modified bases in synthetic mRNAs is an innovation that has allowed for both an ameliorated immune response to the synthetic molecules and increased protein expression from the mRNA, thereby providing an unprecedented opportunity to use synthetic mRNAs as a novel class of therapeutics for a wide range of indications, including two approved vaccines against COVID- . It has been shown that the incorporation of pseudouridine (ψ), N 1 -methyl-pseudouridine (m1ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A) and 2-thiouridine (s2U) into synthetic mRNAs results in reduced immune responses and increased protein expression in vivo [bib_ref] Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance..., Anderson [/bib_ref] [bib_ref] Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Anderson [/bib_ref] [bib_ref] N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced..., Andries [/bib_ref] [bib_ref] A facile method for the removal of dsRNA contaminant from in vitro-transcribed..., Baiersdorfer [/bib_ref] [bib_ref] Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification..., Kariko [/bib_ref] [bib_ref] Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational..., Kariko [/bib_ref] [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref] [bib_ref] N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Parr [/bib_ref] [bib_ref] N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing..., Svitkin [/bib_ref]. Pseudouridine-modified mRNAs have been shown to result in reduced activation of 2′-5′-oligoadenylate synthetase (OAS), RNA-dependent protein kinase (PKR), and toll-like receptors [bib_ref] Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance..., Anderson [/bib_ref] [bib_ref] Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Anderson [/bib_ref] [bib_ref] Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification..., Kariko [/bib_ref]. Investigation of ψ derivatives with improved pharmacological properties led to the identification of m1ψ, the current benchmark for synthetic mRNA-based applications. m1ψ is a naturally occurring modification found in 18S rRNA and tRNAs [bib_ref] RNA nucleotide methylation, Motorin [/bib_ref] [bib_ref] Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal..., Wurm [/bib_ref] [bib_ref] The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific..., Wurm [/bib_ref] , and similar to ψ-modified synthetic mRNAs, the presence of m1ψ in synthetic mRNAs has been demonstrated to result in reduced activation of RNA sensors in cells [bib_ref] N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced..., Andries [/bib_ref] [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref] [bib_ref] N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing..., Svitkin [/bib_ref] [bib_ref] Optimizing modified mRNA in vitro synthesis protocol for heart gene therapy, Hadas [/bib_ref]. Furthermore, the presence of m1ψ in synthetic mRNAs show increased translation efficiency in cell-free extracts, multiple mammalian cell lines, and mouse models [bib_ref] N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced..., Andries [/bib_ref] [bib_ref] A facile method for the removal of dsRNA contaminant from in vitro-transcribed..., Baiersdorfer [/bib_ref] [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref] [bib_ref] N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Parr [/bib_ref] [bib_ref] N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing..., Svitkin [/bib_ref] [bib_ref] Optimizing modified mRNA in vitro synthesis protocol for heart gene therapy, Hadas [/bib_ref] [bib_ref] SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness, Corbett [/bib_ref] [bib_ref] Human 5' UTR design and variant effect prediction from a massively parallel..., Sample [/bib_ref]. The exact mechanism of how m1ψ enhances translation is not well understood but it has been demonstrated that presence of m1ψ, alone or in combination with other chemical modifications, can alter ribosome transit time on the modified mRNA, and can increase the mRNA half-life by altering the secondary structure of the synthetic mRNA [bib_ref] Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Anderson [/bib_ref] [bib_ref] N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing..., Svitkin [/bib_ref] [bib_ref] mRNA structure regulates protein expression through changes in functional half-life, Mauger [/bib_ref] [bib_ref] Pseudouridinylation of mRNA coding sequences alters translation, Eyler [/bib_ref]. Current methods to incorporate modified nucleotides in synthetic mRNAs include complete substitution of the standard nucleotide with a chemically modified nucleotide during the process of in vitro transcription (IVT) by single-subunit DNA-dependent RNA polymerases (ssRNAPs; such as T7, T3, and SP6 RNAP) [bib_ref] mRNA-based therapeutics-Developing a new class of drugs, Sahin [/bib_ref]. In contrast to endogenous mRNAs, in which modified nucleotides occur in specific positions in the mRNA [bib_ref] Dynamic RNA modifications in gene expression regulation, Roundtree [/bib_ref] [bib_ref] Effects of mRNA modifications on translation: An overview, Roy [/bib_ref] , in synthetic mRNAs, the modified nucleotide is present at almost every position where the naturally occurring nucleobase would be. This complete substitution approach is preferred from a regulatory perspective because it results in less molecule-to-molecule variation in the positions of the modified nucleotides along the synthetic mRNA. The exact implications of incorporating the chemical modifications throughout the body of the mRNA is still under investigation. For attaining the maximum effectiveness from the drug product and to ensure that the expression from the synthetic molecule is optimal, it is critical that the modified nucleotide is incorporated in the right place, be compatible with the functional elements of the mRNA, and does not alter the biological function of the synthetic mRNA. Numerous studies have demonstrated that ssRNAPs can incorporate chemically modified nucleotides into RNA 7,10,25 , but it is unclear whether all ss-RNAPs incorporate the modified nucleotides with comparable fidelity. We previously established a Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing-based assay to determine the combined transcription and reverse transcription errors and the effects of RNA modifications [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. In the previous study, T7 RNAP exhibited higher combined error rates in the synthesis of ψ-, m6A-and 5-hydroxymethylcytidine (hm5C)-modified RNAs, with increased misincorporation of ψ across from dT templated bases. In light of the widespread use of m1ψ for mRNA vaccines, in the current study, we focused on understanding the fidelity of incorporation of m1ψ during in vitro transcription using multiple ssRNAPs. The fidelity with which m1ψ is incorporated during in vitro transcription is a timely question to address because both the Spik-eVax (mRNA-1273) and Comirnaty (BNT162b2) SARS-CoV2 mRNA vaccines are synthesized by substituting uridine with m1ψ throughout the body of the mRNA and both have demonstrated efficacy greater than 94% 4,5 . We investigated the fidelity of m1ψ incorporation with three commonly used ssRNAPs (T7, T3 and SP6 RNAPs) and compared it to the fidelity with which uridine and ψ are incorporated. Using four different RNA sequences, including two functional mRNA sequences, we analyzed the effects of sequence context in promoting misincorporation of uridine analogs and identified rA → rU substitution errors as the predominant error when uridine analogs (m1ψ and ψ) are present in the reaction. Finally, based on the nature of the substitution errors observed, we combine sequence optimization of the synthetic mRNA together with an altered RNA synthesis process to reduce the uridine analog incorporation error during in vitro transcription. These proof-of-concept experiments establish a novel method to synthesize mRNAs with improved fidelity of uridine analog incorporation and provide considerations for choosing ssRNAPs for the generation of modified nucleotide-containing mRNAs in vitro. # Results T7 and SP6 RNAPs incorporate m1ψ efficiently. The efficiency of m1ψ incorporation during in vitro transcription was investigated in four different RNA substrates of varying length and sequence. The base composition of the synthesized RNA was analyzed with ultra-high performance liquid chromatography coupled with mass spectrometry. For the long synthetic RNAs with length ranging from 1122 to 4178 nucleotides, the integrity of the RNA was determined using Bioanalyzer. Synthesis of full-length RNAs of expected sizes were observed in reactions performed in the presence of m1ψ with both T7 RNAP and SP6 RNAP . Furthermore, similar to ψ, total m1ψ TP incorporation was as expected in RNA sequences RNA1 (a 1122 nucleotide synthetic sequence that includes all possible four-base combinations) [bib_ref] Examining sources of error in PCR by single-molecule sequencing, Potapov [/bib_ref] and Cypridina luciferase mRNA sequence (CLuc mRNA) when in vitro transcription was performed with T7 RNAP . In order to determine that the RNA synthesized in presence of m1 m1ψTP is indeed of expected length, we subjected two short RNAs, 30-nucleotide RNA and 60-nucleotide long, to intact mass spectrometry analyses [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref]. Irrespective of which uridine analog was present in the reaction, the mass of the predominant species observed in reactions performed with T7 RNAP correspond to the run-off transcripts and few non-templated additions were observed [bib_ref] Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates, Milligan [/bib_ref] suggesting that similar to ψ and uridine, m1ψ is incorporated efficiently during in vitro transcription and the modifications did not disrupt the synthesis of full-length run-off products. m1ψ is incorporated with higher fidelity than ψ by T7 RNAP. To determine the fidelity of m1ψ incorporation during in vitro transcription, we adapted the Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing-based assay to the PacBio Sequel I system to enable higher sequencing capacity and multiplexing [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. We analyzed the errors in the first strand that stem from combined RNAP and RT error, referred hereafter as combined errors. We first determined the combined errors in two synthetic sequences (RNA1 and RNA2) that represent all possible four-base combinations (templates described as DNA-1 and DNA-2 previously [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. The error rates of pooled RNA1 and RNA2 (referred as RNA1/RNA2) in reactions with canonical uridine, using the Sequel I system was observed to be 6.4 ± 0.4 × 10 −5 error/base as compared to 5.6 ± 0.8 × 10 −5 error/base using the PacBio RSII system (Supplementary [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. Since the combined error rates were comparable between the two platforms, we used the Sequel I system for the subsequent experiments. We next tested the error rates in reactions with m1ψ for the two artificial RNA sequences (RNA1 and RNA2), CLuc mRNA as well as BNT162b2 sequences when in vitro transcription reactions are performed with T7 RNAP under standard high-yield reaction conditions. The combined error rates in ψ-modified RNAs were observed to be two-fold higher than unmodified RNAs (1.3 ± 0.2 × 10 −4 errors/base for ψ-containing reactions), while that in m1ψ-modified RNAs were comparable to unmodified RNAs (7.4 ± 0.7 × 10 −5 error/base for m1ψ-containing reactions) and . In order to understand whether the nature of the errors that are introduced when the reactions are performed with different uridine analogs is similar, we compared the error profile observed in the four RNA sequences. Irrespective of the nature of the uridine analog used in the reaction, base substitution was observed to account for the predominant errors ranging from 86 to 95% of total errors for all four RNA sequences www.nature.com/scientificreports/ m1ψ is present in the reaction, we analyzed the substitution profile and Supplementary . For comparing the substitution error profiles, RNA1 and RNA2 data was pooled together to represent substitution errors from artificial RNA sequences and CLuc and BNT162b2 were analyzed separately to understand the substitution error profile of functional mRNAs. For unmodified RNA, no distinct substitution errors were observed for any of the sequences and Supplementary . In contrast, a significant increase in rA → rU/dT → dA www.nature.com/scientificreports/ substitutions was observed when reactions were performed with either m1ψ or ψ and Supplementary Table 2), likely due to rm1ψTP incorporated in place of rATP (opposite dT) by T7 RNA polymerase as it is less likely that m1ψ would have a global effect on the incorporation of other bases. The rA → rU/dT → dA error rates in ψ-modified RNAs were about nine-to 12-fold higher than unmodified RNAs. The rA → rU/dT → dA substitution was also apparent (up to three-fold greater than unmodified RNAs) in m1ψ-modified RNAs but was observed to be less compared to ψ-modified counterpart and Supplementary . Even though the nature of the base substitution errors were similar for all four RNA sequences, we further investigated if there is a specific sequence context within these RNA sequences that have a higher propensity of association with the rA → rU/dT → dA substitution errors observed. Neighboring sequence analyses of the rA → rU/dT → dA sites demonstrated a pronounced 5' rC sequence to be present when the rA → rU/dT → dA substitution was observed in the ψ-modified RNAs [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref] and [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. Furthermore, the rA → rU sites were observed to be distributed throughout the RNA and specific hot spots for the substitutions were not observed for any of the RNA sequences, suggesting that the errors observed do not have a sequence context dependency [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref] and [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. SP6 RNAP incorporates m1ψ with higher fidelity than ψ; overall error rates in reactions performed with SP6 RNAPs are higher than those performed with T7 RNAP. We next sought to compare different RNAPs to determine if m1ψ is incorporated with varied fidelity by different ssRNAPs and if the differences observed in error rates in ψ-and m1ψ-containing RNAs synthesized with T7 RNAP are also observed with other ssRNAPs, T3 and SP6 RNAPs. SP6 RNAP shares 32% identity to T7 RNAP and is also used for generating synthetic mRNAs for therapeutic applications [bib_ref] In vitro transcription of long RNA containing modified nucleosides, Pardi [/bib_ref]. On the other hand, T3 RNAP is 82% identical to T7 RNAP. The total combined error rates observed in reactions performed with T3 RNAP were comparable to reactions performed with T7 RNAP suggesting that these two closely related RNAPs have similar fidelity profile (Supplementary . On the other hand, comparison of the total combined error rates observed from modified and unmodified RNAs synthesized with SP6 RNAP demonstrated one-fold to two-fold higher error rates than those observed with T7 RNAP (Figs. 1a and 3a; . For ψ-modified sequences, the error rate was observed to be 3.3 ± 0.2 × 10 −4 errors/base. The total combined error rates of m1ψ-containing RNAs were 2.5 ± 0 × 10 −4 errors/base. Similar to T7 RNAP, combined error rates followed the same trend-ψ-modified RNAs demonstrating highest error rates as compared to m1ψ-modified RNAs and uridine-containing RNAs. (Figs. 1a and 3a; . Similar to T7 RNAPs, base substitution errors were observed to be the most predominant error type ranging between 89 and 94% of total errors when reactions were performed with SP6 RNAP and Supplementary . In unmodified RNAs synthesized with SP6 RNAPs, the rA → rG/dT → dC substitution was observed to be the predominant error and . However, the substitution profiles of m1ψ-or ψ-modified RNA demonstrated a preponderance of rA → rU/dT → dA substitutions as observed with m1ψ-or ψ-modified RNAs synthesized with T7 RNAP and Supplementary . Compared to the error rates of unmodified RNA, ψ-modified RNAs had seven-to nine-fold increase in rA → rU/dT → dA substitution (Supplementary . For the m1ψ-modified RNAs, the combined error rates were two-to three-fold higher than unmodified RNA. Furthermore, the sequence context analysis of the rA → rU substitutions observed in RNAs synthesized with SP6 RNAP demonstrated enrichment of a 5' rC for both unmodified and modified RNAs . Additionally, the substitution sites were distributed throughout the length of the transcript as observed in RNAs synthesized with T7 RNAP . Taken together, SP6 RNAP has a higher total error rate as compared to T7 RNAP but the error profile and the distribution of the error is comparable between these RNAPs. www.nature.com/scientificreports/ Fidelity of uridine incorporation is not dependent on the yield of the in vitro transcription reaction. For synthetic mRNA-based applications, high yield of RNA from the in vitro transcription reaction is desirable and reactions are typically performed with high concentrations of rNTPs. The recommended highyield rNTP concentrations are different for T7 RNAP (40 mM rNTP) and SP6 RNAP (20 mM rNTP). In order to ensure that the differences in combined error observed for T7 RNAP and SP6 RNAP are not due to differences in the rNTP concentrations in the reactions, we performed in vitro transcription reactions with T7 RNAP under low rNTP reaction conditions with either 20 mM or 10 mM rNTP (total). The total combined error rates as well as the base substitution errors observed in unmodified RNA1/RNA2 when reactions were performed with T7 RNAP under low rNTP (10 or 20 mM) reaction conditions were comparable to that observed with high rNTP (40 mM) reaction conditions (Supplementary suggesting that the overall rNTP concentration in the reaction does not affect fidelity of uridine incorporation and the differences in error rates observed with T7 and SP6 RNAP are not due to differences in the reaction conditions. Altering the rNTP composition during in vitro transcription reduces combined error rate and the predominant rA-to-rU substitution error. Our data demonstrates that m1ψ-and ψ-modified RNAs have increased combined errors compared to unmodified RNAs and the increased rA → rU substitution errors account for most of the misincorporations that are observed . Furthermore, the increased rA → rU substitution errors observed in RNAs synthesized with SP6 RNAP suggest that the predominant rA → rU substitution might occur during in vitro transcription where the uridine analogs are misincorporated with higher frequency. In order to test this hypothesis as well as to reduce the rA → rU substitution during in vitro transcription reactions with uridine analogs, we decided to manipulate the rUTP concentration in the reaction with the idea that balancing the rUTP in the in vitro transcription reaction to match the nucleotide composition of the RNA sequence might result in reduced rA → rU substitution errors and consequently the total errors observed during in vitro transcription. One aspect of rationalized mRNA design that has been gaining traction has been to reduce the uridine composition without altering the amino acid sequence of the protein encoded from the synthetic mRNA [bib_ref] Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity..., Vaidyanathan [/bib_ref] [bib_ref] Modifications in an emergency: The role of N1-methylpseudouridine in COVID-19 vaccines, Nance [/bib_ref]. Uridinedepletion in Cas9 mRNA sequence demonstrated a reduction of innate immune response and an increase in Cas9 activity [bib_ref] Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity..., Vaidyanathan [/bib_ref]. Comirnaty and Spikevax sequences consist of 19% and 15% uridine, respectively, as compared to the wild-type spike protein sequence that has 33% uridine in the sequence [bib_ref] Modifications in an emergency: The role of N1-methylpseudouridine in COVID-19 vaccines, Nance [/bib_ref]. Often, rationalized design of the synthetic mRNA molecule is further combined with reaction optimization such as altering the rNTP concentrations in the reaction to optimize the RNA yields from the reactions as well as reduction of dsRNA byproducts [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref] [bib_ref] Optimizing modified mRNA in vitro synthesis protocol for heart gene therapy, Hadas [/bib_ref]. First, as a control, we analyzed the error rates when in vitro transcription reactions were performed under standard high-yield rNTP condition where all the rNTPs are added equally to a final concentration of 40 mM (represented as equal in [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref] and Supplementary . The uridine-depleted RNA sequences transcribed with T7 RNAP with equal molar unmodified rNTPs had a total combined error rate 6.0 ± 1.1 × 10 −5 errors/ base, while the error rates of ψ-and m1ψ-incorporating transcripts were observed to be 1.9 ± 0.4 × 10 −4 and 9.9 ± 3.0 × 10 −5 errors/base, respectively [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. For the rUTP optimized reactions, no difference in the RNA yield from reactions with modified uridine analogs was observed (Supplementary . Furthermore, the integrity of the RNA was not compromised when reactions were performed with proportional rNTPs [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. The total combined error rate of unmodified RNAs was observed to be 5.6 ± 2.1 × 10 −5 errors/base and that of m1ψ-incorporating RNAs was 6.3 ± 0.5 × 10 −5 errors/base . Noticeably, the total error rate of ψ-modified RNAs was 8.4 ± 0.9 × 10 −5 errors/base, about one-fold reduced as compared to equal molar rNTP condition (1.9 ± 0.4 × 10 −4 errors/base). Under standard equal molar rNTP reaction conditions, the substitution error profile of the uridine-depleted RNA sequences resembled those of RNA1/RNA2, CLuc mRNA and BNT162b2, with rA → rU/dT → dA substitution demonstrating the most significant change when modified uridine was used in the reaction (Figs. 1c, 4c,d; ; Supplementary Tables 9 and 10). For the RNA sequence with 5.5% uridine content, the rA → rU/dT → dA substitution was increased three-fold in m1ψ-modified RNA as compared to the unmodified RNA and this was even more pronounced in the ψ-modified RNA with an increase of 14-fold over unmodified RNA. Interestingly, when the rNTP concentrations were altered to match the nucleotide content of the sequence, the rA → rU substitution error rates were lowered significantly as compared to that observed under standard reaction condition (equal rNTP conditions). For unmodified RNA, a four-fold reduction in rA → rU substitution was observed when rUTP concentration was altered to match the template sequence [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref] and Supplementary . Similarly, six-and seven-fold reductions were observed for ψ-modified and m1ψ-modified RNA, respectively. Furthermore, no significant change in any of the other substitution errors were observed when the rNTP concentrations were altered [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. Similar fold changes were also observed for two other artificial RNA sequences with uridine content of 6.6% or 12.3% and U-depleted CLuc mRNA sequence [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref] ; . The comparison of the two different RNA synthesis workflows with uridine-depleted RNA sequences demonstrates that lowering the rUTP concentration to match the nucleotide sequence of the RNA indeed reduced the rA → rU substitution error during in vitro transcription reaction and further provides support that the rA → rU substitutions observed with the uridine modifications are errors observed during in vitro transcription. In light of our observation that the fidelity of T7 RNAP can be modulated by adjusting the rNTP ratio in the reaction, we investigated if the same holds true for SP6 RNAP where the rA → rU substitution was also observed to be the most predominant substitution error in modified RNAs. We used the same T-depleted randomized sequence template (resulting in 5.5% uridine content in the RNA) and reactions were performed with either equimolar rNTPs or rNTPs matched to the nucleotide sequence. In presence of proportional rNTPs, the total combined error rate in unmodified RNA was reduced to 1.3 ± 0 × 10 −4 errors/base, one-fold decrease from . Similarly, for m1ψ-modified reaction, the total combined error rate was reduced two-fold (from 4.1 ± 1.0 × 10 −4 errors/base to 1.5 ± 0 × 10 −4 errors/base). As observed with T7 RNAP, ψ-modified RNA had the most pronounced (threefold) reduction in total combined error rate (from 6.0 ± 2.1 × 10 −4 errors/base to 1.7 ± 0 × 10 −4 errors/base) when the rNTP ratios were altered to match the sequence of the RNA. Furthermore, the reduction in total combined error observed in proportional rNTP reaction conditions is attributable specifically to reduction in the rA → rU substitution errors and . Uridine-depletion of the RNA sequence is not a viable alternative for all sequences. Furthermore, the extent of uridine-depletion is dependent on the sequence since it requires change in the uridine content without altering the codon. We investigated a corollary approach where we hypothesized that increasing the rATP concentrations in the reaction might also reduce the rA → rU substitutions in the in vitro transcription reactions. Altering the rNTP ratio did not compromise the integrity of the full-length transcript . When 16 mM rATP was used in the reactions (with 8 mM each of the other three rNTPs), the total combined error rates of unmodified, ψand m1ψ-modified RNA1/RNA2 were observed to be 4.5 ± 0.5 × 10 −5 errors/base, 6.4 ± 0 × 10 −5 errors/base and 6.0 ± 0.9 × 10 −5 errors/base, respectively (Supplementary . When 20 mM rATP was used (with 10 mM each of the other three rNTPs), the total combined error rates of unmodified, ψ-and m1ψ-modified RNA1/ RNA2 were observed to be 4.7 ± 0 × 10 −5 errors/base, 6.6 ± 0.2 × 10 −5 errors/base and 4.8 ± 0.7 × 10 −5 errors/base, ; a two-fold decrease in total combined error rates for ψ-modified RNAs. As hypothesized, the substitution error profile of reactions performed with excess rATP showed rA → rU/dT → dA substitution in the modified RNA changed the most compared to unmodified RNA and . Taken together, these results demonstrate that the rA → rU substitution that is observed when in vitro transcription reactions are performed with uridine analogs, stems from substitution errors during in vitro transcription with T7 and SP6 RNAPs. Furthermore, the fidelity of the uridine analog incorporation can be increased by either lowering the rUTP concentration to match the nucleotide composition of the synthetic RNA sequence or increasing the rATP concentration in the reaction without compromising the yield from the reaction. # Discussion The current approaches to introduce modifications in synthetic mRNAs involve replacing a canonical nucleotide with a modified analog during in vitro transcription by ssRNAPs. T7 and SP6 RNAPs are the most commonly used RNA polymerases for in vitro transcription. Both T7 and SP6 have their own promoter specificities and it is also well known that they result in heterogeneous RNA populations [bib_ref] Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates, Milligan [/bib_ref] [bib_ref] Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes..., Melton [/bib_ref] [bib_ref] SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates, Stump [/bib_ref]. However, whether or not these two RNA polymerases incorporate modified nucleotides with similar fidelities has not been tested till date. Our data demonstrates that the combined error rates in RNAs synthesized with SP6 RNAP were up to two-fold higher than those observed with T7 RNAP ; ; [fig_ref] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated... [/fig_ref]. On the other hand, T3 RNAP exhibited error rates comparable to that of T7 (Supplementary . That said, it is interesting to note that the error profiles of uridine-modified RNA for both T7 and SP6 polymerases were similar. For both RNAPs, the incorporation of ψ or m1ψ was more error prone than canonical uridine incorporation (Figs. 1a and 3a; . However, when uridine analogs were present in the reaction, both T7 and SP6 RNAPs had a very similar substitution error profile with rA → rU substitution being the predominant error observed during in vitro transcription (Figs. 1c and 3c; Supplementary Tables 2 and 5). Available crystal structure capturing T7 RNAP during transcription elongation, as well as biochemical assays and molecular dynamics simulation, have identified several key residues that contribute to transcription fidelity [bib_ref] Misincorporation by wild-type and mutant T7 RNA polymerases: Identification of interactions that..., Huang [/bib_ref] [bib_ref] Structural basis for substrate selection by t7 RNA polymerase, Temiakov [/bib_ref] [bib_ref] A critical residue selectively recruits nucleotides for t7 RNA polymerase transcription fidelity..., Duan [/bib_ref]. For example, Tyrosine 639 is known to be critical for the pre-insertion of correct nucleotides opposite to the template DNA strand [bib_ref] A critical residue selectively recruits nucleotides for t7 RNA polymerase transcription fidelity..., Duan [/bib_ref]. Since the differences in the total combined errors between T7 and SP6 RNAPs were observed for the same template sequences, and since base pairing between incoming rNTP and DNA template is the same, the fidelity differences of the two enzymes must be associated with differences in the protein sequence, e.g. residues which function to ensure correct base pairing. Interestingly, Tyr639 is conserved in both T7 and SP6 RNAPs as well as among other bacteriophage ssRNAPs, but neighboring amino acids are not. Future studies to elucidate the role of residues outside of the active site on fidelity of modified nucleotide incorporation will be instrumental in designing high-fidelity in vitro transcription systems. In addition to targeted amino acid replacements in T7 or SP6 RNAP, determining the error rates of homologous ssRNAPs will reveal the sequence determinants of transcriptional fidelity within this protein family. Furthermore, it will also be of interest to identify novel ssRNAPs that have a distinct substitution error profile than that of T7 RNAP and might be a better RNAP candidate for introduction of uridine analogs. Even though the combined error rates observed for the same set of RNA sequences were significantly different between T7 and SP6 RNAPs, there were few similarities. First, the fidelity with which the analogs were incorporated, followed the same trend-uridine > m1ψ > ψ-with m1ψ-modified RNA exhibiting lower total errors and rA → rU/dT → dA substitution than ψ-modified RNA . Second, the difference in fidelity between uridine-containing RNAs and ψ/m1ψ-modified RNAs is mainly attributable to a higher rA → rU/dT → dA substitution error . Based on our data from multiple templates and reaction conditions, we demonstrate that both ψ and m1ψ have a higher propensity to mis-pair with dT in the template DNA during in vitro transcription. Biophysical studies to measure the melting temperatures (T m ) of synthetic RNA duplexes containing either uridine, ψ or m1ψ, have shown that both ψ-or m1ψ-containing duplexes have a higher T m than uridine-containing duplexes [bib_ref] N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Parr [/bib_ref]. Increased base pairing and stacking has been postulated to contribute to the increased Tm observed 30,37-39 . One of the critical features of ψ and m1ψ is the C5-C'1 bond that enables rotation between the sugar moiety and the nucleobase, which, in contrast to canonical uridine, could provide improved base pairing and stacking. As compared to uridine and m1ψ, ψ contains an extra hydrogen bond donor group (N1H) that imparts a universal base character to ψ. Hence, ψ can not only pair with A but can also wobble base-pair with G, U, or C 36 . On the other hand, m1ψ has a methyl group in the N1-position and therefore does not have the extra hydrogen bond donor and therefore wobble pairing with other nucleotides is not favored. It is plausible that the lower error rates observed in m1ψcontaining RNAs is due to the lack of the extra hydrogen bond donor [bib_ref] The critical contribution of pseudouridine to mRNA COVID-19 vaccines, Morais [/bib_ref]. Additionally, it is likely that the methyl group in the N1 position of m1ψ may alter the polarity of the C2 position and make it less favorable to pair with dT in the DNA template. It is also worth noting that stability difference between RNA duplex containing four consecutive ψ's and m1ψ's was not distinguishable 13 . Stability measurements in which a single rψTP or rm1ψTP pairs with DNA template to mimic the errors observed during in vitro transcription will likely be more relevant to distinguish the base-pairing differences we observe in this study. Not much is understood about the pairing of rψ or r m1ψ with DNA, but there is precedence for RNA-RNA interactions in presence of ψ and m1ψ which might help us understand the increased error rates observed in presence of the modified analogs [bib_ref] N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Parr [/bib_ref] [bib_ref] Pseudouridines or how to draw on weak energy differences, Westhof [/bib_ref] [bib_ref] The critical contribution of pseudouridine to mRNA COVID-19 vaccines, Morais [/bib_ref] [bib_ref] The contribution of pseudouridine to stabilities and structure of RNAs, Kierzek [/bib_ref]. Combining sequence optimization of the synthetic RNA with incorporation of uridine modifications, specifically m1ψ and ψ, in synthetic mRNA-based vaccines and therapeutics is becoming a common practice. In addition, depleting the uridine content in the synthetic mRNA by sequence optimization has been demonstrated to reduce the immunogenicity of the synthetic molecules [bib_ref] Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity..., Vaidyanathan [/bib_ref] [bib_ref] Modifications in an emergency: The role of N1-methylpseudouridine in COVID-19 vaccines, Nance [/bib_ref]. Our data demonstrates that combining uridine depletion of the RNA sequence with altering the rNTP composition of the reaction, reduces the rA → rU substitutions [bib_ref] Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification..., Kariko [/bib_ref] [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref]. Future studies aimed at comparing the outcome from these modified processes (uridine depletion with altered rNTP composition to generate high-fidelity synthetic mRNA with reduced dsRNA byproducts) will provide further insights into ways to optimize the efficacy from the synthetic mRNA drug substance. Our observation that the prevalent rA → rU substitutions introduced during in vitro transcription can be reduced by balancing the rNTPs in the reaction suggests a few possible mechanisms for introduction of these substitution errors. By limiting the rψTP amounts or competing with excess rATP, the rA → rU substitution can be altered to reduce the error rates without affecting any of the other substitution errors and Supplementary . One of the possible reasons for this could be, that these mispairing events occur in presence of excess rNTPs in the reaction. This is further supported by the fact that in experiments where the template sequence was intentionally biased to have reduced uridine, adding equal molar rNTPs in the reaction (10 mM each) led to an overall increased error profile than that observed in templates that had comparable nucleotide usage (RNA1 and RNA2). Interestingly, the nucleotide composition of RNA1 and RNA2 have equal representation of all the four nucleotides and we still observed prevalent rA → rU substitutions when equal molar rNTPs were added in the reaction [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref]. These reactions were performed with 10 mM rNTP each. It is possible that not all the rNTPs in the reaction are utilized by T7 RNAP there is always excess of rNTPs in these high-yield rection conditions. Future studies to dissect out if the misincorporations are more prevalent in the early vs late stage of the in vitro transcription process will provide further insight into the events leading to uridine analog misincorporation. It can also be envisioned that in vitro transcription systems that limit the initial rNTP concentration but allow for a steady, optimized rNTP feeding mechanism might further help improve the fidelity of nucleotide incorporation and be instrumental in generating high-fidelity synthetic mRNAs. In order to achieve high-fidelity RNA products, it is desirable to understand the rules of nucleotide incorporation so that if there are sequences that are more error prone, they can be omitted from the synthetic mRNA during design. However, in this study, the comparison of multiple sequence contexts demonstrated that other than T7 RNAP-incorporated ψ-modified RNAs that demonstrated slight sequence context preference, there is no strong correlation between the sequence context of the DNA template and the substitution errors observed under any condition tested [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref]. Furthermore, the errors observed were distributed throughout of the length of RNA [fig_ref] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have... [/fig_ref]. Our results demonstrate that the presence of ψ and m1ψ in the in vitro transcription reactions result in higher base substitution errors in the modified RNAs. A critical aspect to consider is how these errors might affect the efficacy of the synthetic mRNA drug substance and how much of these error-prone molecules can be tolerated in vivo without any adverse effect. Because in vitro transcribed mRNAs are modified throughout the body of the mRNA, it is also critical to consider if these mRNAs are faithfully translated in the cell. We have previously demonstrated that in human embryonic kidney cell, low frequency translation elongation miscoding events are observed from ψ-containing mRNAs due to altered tRNA selection in ψ-containing codons [bib_ref] Pseudouridinylation of mRNA coding sequences alters translation, Eyler [/bib_ref]. For m1ψ-substituted RNAs, it has been shown that translation initiation and ribosome transit is altered in vivo [bib_ref] N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing..., Svitkin [/bib_ref]. A complete understanding of what errors are incorporated during the RNA synthesis process and how that further affects the identity of the protein synthesized is a timely question given that therapeutic applications would require repeat dosing of the mRNAs and would also require expression of the protein of choice. To be able to predict the best outcome from these synthetic molecules, it is critical that we understand where variability comes from and to be able to define the rules to avoid these variabilities. # Materials and methods Oligonucleotides and DNA template sequences used in this study. All of the oligonucleotides for in vitro transcription and reverse transcription were synthesized by Integrated DNA Technologies (IDT, Coralville IA) and the sequences are available in . The DNA template sequences for in vitro transcription are available in . ## Generation of dna templates for in vitro transcription (ivt) of long rnas. generation of the DNA templates for the artificial RNA sequences RNA1 and RNA2 (1122-and 1124-nucleotide sequence that includes all possible four-base combinations) were described earlier [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. For in vitro transcription reactions with SP6 or T3 RNA polymerase, the corresponding promoter sequences were inserted in the DNA templates using Q5 Site-Directed Mutagenesis Kit (E0554, New England Biolabs). DNA templates encoding functional mRNAs, Cypridina luciferase (CLuc) mRNA (1707 nucleotides) and part of BNT162b2/Comirnaty mRNA (4187 nucleotides) [bib_ref] Modifications in an emergency: The role of N1-methylpseudouridine in COVID-19 vaccines, Nance [/bib_ref] , were synthesized by GenScript Inc. (GenScript, Piscataway NJ) and introduced into standard high-copy plasmids. DNA templates for uridine-depleted artificial sequences (with 5.5%-, 6.6%-and 12.3%-T content) were synthesized by GenScript and introduced into standard high-copy plasmids. The plasmids were propagated in E. coli (C2987, New England Biolabs) and purified with the Monarch Plasmid Miniprep Kit (T1010, New England Biolabs). T-depleted (13.5% T-content) version of the Cypridina luciferase (CLuc) mRNA template was synthesized by GenScript and introduced into standard high-copy plasmids. The plasmids were propagated in E. coli (C2987, New England Biolabs) and purified with the Monarch Plasmid Miniprep Kit (T1010, New England Biolabs). Plasmids were digested with restriction enzymes to generate linearized templates for in vitro transcription. The linearized plasmids were treated with PreCR Repair Mix (M0309, New England Biolabs) and purified with the Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs). For short RNAs (30-nucleotide and 60-nucleotide long), double-stranded DNA templates were generated by annealing DNA oligonucleotides for template and non-template strands in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA) by heating at 95 °C for 5 min followed by gradual cooling to room temperature. www.nature.com/scientificreports/ DNA templates corresponding to three uridine-depleted artificial RNA sequences with base composition of (1) 30.9% A, 33.4% C, 30.1% G, 5.5% U, (2) 29.7% A, 32.0% C, 31.7% G, 6.6% U and (3) 30.2% A, 30.4% C, 27.1% G, 12.3% U were synthesized by GenScript Inc. (GenScript, Piscataway NJ). Additionally, to evaluate if this approach is viable for a functional mRNA, we also generated a uridine-depleted CLuc mRNA sequence where the uridine content was reduced from 22.8 to 13.5%. The uridine-depletion of the CLuc mRNA sequence was performed without perturbing the amino acid sequence of the encoded protein so that there are no other differences between the two mRNAs, other than the nucleotide content of the sequences . In vitro transcription (IVT). In vitro transcription reactions were performed according to standard protocols provided with the high-yield in vitro transcription kits (E2040 and E2070, New England Biolabs), consisting of 40 mM rNTP (pH buffered with sodium phosphate) for T7 RNA polymerase and 20 mM rNTP (pH buffered with Tris) for SP6 RNA polymerase. In vitro transcription with T3 RNA polymerase (M0378S, New England Biolabs) was carried out following manufacturer's instruction. For modified RNAs, UTP was replaced with either pseudouridine-5'-triphosphate (N-1019, TriLink Biotechnologies) or N 1 -Methylpseudouridine-5'-Triphosphate (N-1081, TriLink Biotechnologies). For the uridine-depleted sequences (RNA with 5.5%, 6.6% and 12.3% uridine content), two sets of reactions were performed-one in which all the rNTPs were present in equimolar ratio and another in which the rNTPs were altered to match the template sequence composition. Following IVT, the DNA template was digested with Turbo DNase (AM2238, Invitrogen) digestion at 37 °C for 30 min and then purified with the Monarch RNA Cleanup Kit (T2050, New England Biolabs) for long RNA (1020 nucleotides to 4187 nucleotides) or with the Oligo Clean-Up and Concentration Kit (34100, Norgen Biotek Inc) for short 30-nt RNA and 60-nt RNA. Low-yield IVT reaction conditions were carried out with either 20 mM or 10 mM rNTPs (total) at 37 °C for two hours. DNA template removal and cleanup were performed as described above. For the rUTP optimized T7 RNAP reactions, rNTPs proportional to the template sequence were used [for e.g., 12.4 mM (31%) rATP, 13.2 mM (33%) rCTP, 12 mM (30%) rGTP and 2.4 mM (6%) rUTP was used for RNA sequence with 5.5% uridine content]. For the rUTP optimized SP6 RNAP reactions, rNTPs proportional to the template sequence were used (for e.g., 6.2 mM (31%) rATP, 6.6 mM (33%) rCTP, 6 mM (30%) rGTP and 1.2 mM (6%) rUTP was used for RNA sequence with 5.5% uridine content]. In vitro transcription of RNA1/RNA2 with excess rATPs were performed with either 16 mM rATP with 8 mM of other rNTPs or 20 mM rATP with 10 mM of other rNTPs. Bioanalyzer for RNA size distribution and integrity. Eluted RNA samples from the in vitro transcription reactions were diluted based on concentrations measured on a Nanodrop spectrophotometer (13-400-519, Thermo Fisher Scientific) and denatured at 70 °C for 2 min and snap-cooled on ice. 250 ng RNA samples were analyzed with RNA 6000 Nano kits (5067, Agilent Technologies) and the integrity and size distribution of the RNA was assessed using mRNA Nano series 2 assay (G2938, Agilent Technologies). Nucleoside digestion of RNA and UHPLC-MS analyses to assess modification incorporation. Purified modified RNA and RNA without any chemical modification were digested with the Nucleoside digestion mix (M0649, New England Biolabs) at 37 °C for 1 h. Base composition analysis was performed by Liquid Chromatography-Mass Spectrometry (LC-MS) using an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135XT MS Detector, on a Waters Xselect HSS T3 XP column (2.1 × 100 mm, 2.5 µm) with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5). Intact mass spectroscopy analysis for short RNA oligonucleotides. Purified 30-nucleotide and 60-nucleotide RNA with different uridine modifications were analyzed at Novatia, LLC using on-line desalting, flow injection electrospray ionization on an LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific) and analyzed with ProMass Deconvolution software from Novatia, LLC (https:// www. enova tia. com/ our-produ cts/ proma ss/? gclid= EAIaI QobCh MIoZS 6xKDn-AIViN rICh3 peATr EAAYA SAAEg LRNPD_ BwE). First and second strand cDNA synthesis for PacBio. The cDNA synthesis was performed as described before with a modified cleanup step using Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs) [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. Sequences of forward and reverse oligonucleotides used are provided in the . Pacific biosciences SMRTbell library preparation and sequencing. The library preparation for sequencing on RSII system was performed as described before [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. For the sequencing on the Sequel platform, about 1.5 µg cDNA was treated with NEBNext End Repair Module (E6050, New England Biolabs) at room temperature for 5 min, followed by purification with Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs). The end-repaired cDNA was ligated with 2 µL barcoded adaptor (100-466-000, Pacific Biosciences) with T4 DNA Ligase (M0202, New England Biolabs) in 50 µL reaction volume at room temperature for [bib_ref] mRNA-based therapeutics-Developing a new class of drugs, Sahin [/bib_ref] www.nature.com/scientificreports/ protocol for primer annealing, polymerase binding [Sequel Binding Kit 3.0 (101-613-900, Pacific Biosciences)], cleanup and final loading to SMRT Cells LR and sequencing using Sequel system. Data analysis. Analysis of sequencing data was performed as previously described [bib_ref] Base modifications affecting RNA polymerase and reverse transcriptase fidelity, Potapov [/bib_ref]. In short, high-accuracy consensus sequences were built for the first and second strand for each sequenced double-stranded DNA. The consensus sequences were aligned to the reference sequence and base substitutions, deletions, and insertions were determined. The first strand error rates were determined by comparing the first strand consensus sequences to the reference sequence (RNA strand), and mutations were required to be present in both strands. The average error and standard deviation of total errors were then calculated by combining the data from the templates for each reaction condition: for RNA1 and RNA2, one measurement was performed and for other RNA sequences, two independent repeats were performed. Substitution error profiles are represented separately for each RNA sequence. The relative fold change was calculated for each substitution as (M − U)/U, where M is the substitution rate on modified RNA (m1ψ or ψ) and U is the substitution rate on unmodified RNA. For comparing total combined error rates and for estimating the statistical significances, p-values were calculated using unpaired t-tests from the measurements of unmodified and modified RNAs, and those under equal and proportional rNTP conditions. The local context of base substitutions was extracted, and sequence logos were built using WebLogo software [bib_ref] A sequence logo generator, Crooks [/bib_ref]. Also, the frequency of mutations at each position was plotted to examine distribution of errors along the reference sequence. ## Data availability Sequencing data has been deposited into the Sequencing Read Archive (https:// www. ncbi. nlm. nih. gov/ sra) under accession number SRP375186. Received: 6 June 2022; Accepted: 22 July 2022 [fig] Figure 2: rA → rU/dT → dA substitution errors observed in ψ-containing RNAs have sequence context preference and occur throughout the transcript body when reactions are performed with T7 RNA polymerase. The sequence context preference and distribution of the rA → rU/dT → dA substitution errors observed in CLuc and BNT162b2 mRNAs are represented. (a) Logo of sequence surrounding the rA → rU/dT → dA substitution sites. Seven nucleotides upstream and downstream of the substitution site were examined and for simplicity the immediate sequence upstream and downstream of the substitution site is represented. Sequence logos were built using WebLogo software41 and the numbers of sites plotted are summarized in Supplementary Table 15. (b) Substitution error distribution along the length of the transcript. Scientific Reports | (2022) 12:13017 | https://doi.org/10.1038/s41598-022-17249-1www.nature.com/scientificreports/ RNA (three different sequences), the total combined error rates observed when reactions were performed with SP6 RNAP under standard high-yield reaction conditions were greater than that of T7 RNAP with combined error rates of 1.3 ± 0 × 10 −4 errors/base,(Fig. 3aand Supplementary [/fig] [fig] Figure 4: In vitro transcription error profile of T7 RNA polymerase can be modulated by altering ribonucleotide composition in the reaction. Uridine-depleted artificial RNA sequences and uridine-depleted CLuc mRNA were transcribed with T7 RNA polymerase and reactions were performed with either equal molar rNTPs or rNTPs proportional to the template sequence composition. (a) Combined first-strand errors are an average of four uridine depleted RNA sequences under the two different rNTP conditions. The three synthetic RNAs have uridine content of 5.5%, 6.6% and 12.3%; CLuc mRNA has a uridine content of 13.5%. Unpaired t-tests were used to calculate p-values. (b) Bioanalyzer traces of synthetic RNA with 5.5% uridine content demonstrating synthesis of full-length IVT products under both reaction conditions. (c) Base substitution error profile observed for unmodified and modified uridine-depleted artificial RNA sequences (RNA with 5.5% uridine content) under two different rNTP reaction conditions. (d) Base substitution error profile observed for unmodified and modified uridine-depleted CLuc mRNA from under the two different rNTP reaction conditions. Colors indicate specific substitution errors observed. [/fig] [fig] Figure 5, Figure 6: In vitro transcription error profile of SP6 RNA polymerase can be modulated by altering ribonucleotide composition in the reaction. Uridine-depleted artificial RNA sequence (uridine content of the sequence is 5.5%) was transcribed with SP6 RNA polymerase and reactions were performed with either equal molar rNTPs or rNTPs proportional to the template sequence composition. Base substitution error profile observed for unmodified and modified uridine-depleted RNA sequence under the two different rNTP reaction conditions. Colors indicate specific substitution errors observed. In vitro transcription error profile of T7 RNA polymerase can be modulated by increasing the rATP concentration in the reaction. Artificial RNA sequences RNA1 and RNA2 were transcribed with T7 RNA polymerase and reactions were performed with excess of rATP(16 mM or 20 mM) as compared to the other rNTPs in the reaction (8 mM or 10 mM respectively). (a) Bioanalyzer traces of RNA1 synthesized with either 16 mM or 20 mM rATP demonstrating synthesis of full-length IVT products. (b) Base substitution error profile observed for unmodified and modified RNA sequences under the different rATP concentrations. [/fig]

The human DEAD-box helicase DDX3X as a regulator of mRNA translation The human DEAD-box protein DDX3X is an RNA remodelling enzyme that has been implicated in various aspects of RNA metabolism. In addition, like many DEAD-box proteins, it has non-conventional functions that are independent of its enzymatic activity, e.g., DDX3X acts as an adaptor molecule in innate immune signalling pathways. DDX3X has been linked to several human diseases. For example, somatic mutations in DDX3X were identified in various human cancers, and de novo germline mutations cause a neurodevelopmental condition now termed 'DDX3X syndrome'. DDX3X is also an important host factor in many different viral infections, where it can have pro-or anti-viral effects depending on the specific virus. The regulation of translation initiation for specific mRNA transcripts is likely a central cellular function of DDX3X, yet many questions regarding its exact targets and mechanisms of action remain unanswered. In this review, we explore the current knowledge about DDX3X's physiological RNA targets and summarise its interactions with the translation machinery. A role for DDX3X in translational reprogramming during cellular stress is emerging, where it may be involved in the regulation of stress granule formation and in mediating non-canonical translation initiation. Finally, we also discuss the role of DDX3X-mediated translation regulation during viral infections. Dysregulation of DDX3X's function in mRNA translation likely contributes to its involvement in disease pathophysiology. Thus, a better understanding of its exact mechanisms for regulating translation of specific mRNA targets is important, so that we can potentially develop therapeutic strategies for overcoming the negative effects of its dysregulation. Ryan CS and Schröder M (2022), The human DEAD-box helicase DDX3X as a regulator of mRNA translation. # Introduction The human DEAD-box protein 3X (DDX3X) is a multifunctional protein implicated in a variety of biological processes. Paralogue DDX3 genes are located on the X and Y chromosomes, giving rise to two closely related proteins: DDX3X and DDX3Y [bib_ref] Assignment of a human putative RNA helicase gene, DDX3, to human X..., Park [/bib_ref] [bib_ref] Gene structure of the human DDX3 and chromosome mapping of its related..., Kim [/bib_ref]. DDX3X is expressed in both males and females and can escape X-chromosome inactivation in the latter [bib_ref] Landscape of DNA methylation on the X chromosome reflects CpG density, functional..., Cotton [/bib_ref]. DDX3Y is expressed only in males and displays some functional redundancy with DDX3X, however it also has a different tissue expression pattern, which may have implications for sex differences in DDX3-related function and disease. Although its expression was thought to be posttranscriptionally restricted to male germ cells, DDX3Y has also been implicated in neurodevelopment and immune cell function, suggesting that it may take part in physiological processes outside of spermatogenesis [bib_ref] DDX3Y, a male-specific region of Y chromosome gene, may modulate neuronal differentiation, Vakilian [/bib_ref] [bib_ref] The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity, Szappanos [/bib_ref]. In this review, we focus on DDX3X, as it is the better studied of the two paralogues. However, it is possible that DDX3Y makes a significant contribution to overall DDX3 function in males, which should be considered in future studies to more clearly delineate redundancies and separate functions of the two paralogues. It is also possible that DDX3Y contributes to functional effects attributed to DDX3X in some studies discussed here, depending on cell types and the extent of cross-reactivity of research tools. DDX3 orthologues in other organisms include Ded1 in Saccharomyces cerevisiae [bib_ref] Biochemical differences and similarities between the DEAD-box helicase orthologs DDX3X and Ded1p, Sharma [/bib_ref] and Belle in Drosophila melanogaster, and we include some results relating to these well-studied orthologues in this review. DDX3X and its orthologues are members of the DEAD-box protein family of RNA remodelling enzymes. As such, DDX3X contains two conserved RecA-like domains that constitute its helicase core, which are flanked by N-and C-terminal extensions unique to DDX3X . The extended N-and C-termini of DDX3X contain intrinsically disordered regions that confer much of DDX3X's unique functionality by mediating many different protein-protein interactions and by conferring a propensity for liquid-liquid phase separation [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref] [bib_ref] Acetylation of intrinsically disordered regions regulates phase separation, Saito [/bib_ref]. As an RNA helicase, DDX3X hydrolyses ATP to ADP and thereby destabilizes secondary structures of bound RNAs [bib_ref] Biochemical differences and similarities between the DEAD-box helicase orthologs DDX3X and Ded1p, Sharma [/bib_ref] [bib_ref] HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity, De Bisschop [/bib_ref]. This RNA remodelling activity allows DDX3X to have numerous functions related to RNA metabolism, being implicated in transcription [bib_ref] DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional..., Chao [/bib_ref] [bib_ref] The RNA helicase DDX3 promotes IFNB transcription via enhancing IRF-3/p300 holocomplex binding..., Saikruang [/bib_ref] mRNA export [bib_ref] Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function, Yedavalli [/bib_ref] [bib_ref] The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well..., Lai [/bib_ref] [bib_ref] HIV-1 recruits UPF1 but excludes UPF2 to promote nucleocytoplasmic export of the..., Ajamian [/bib_ref] , and translation. Of note, DDX3X also has biological functions that do not require its ATPase/helicase activity. For example, it acts as an innate immune signaling adaptor in the RIG-I pathway, where it bridges interactions between various signaling proteins to promote induction of type I interferon [bib_ref] Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated..., Schröder [/bib_ref] [bib_ref] Human DEAD box helicase 3 couples IκB kinase ε to interferon regulatory..., Gu [/bib_ref]. A similar adaptor function has also been shown for its involvement in the WNT pathway [bib_ref] RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in..., Cruciat [/bib_ref]. Additionally, DDX3X is a positive regulator of the NLRP3 inflammasome and as such contributes to inflammation and pyroptosis [bib_ref] DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3..., Samir [/bib_ref]. Thus, DDX3X is a multifunctional protein, and some of its roles fall outside its ability to remodel RNA. Here, we focus on DDX3X's ability to regulate translation, as this is an intensively investigated aspect of its function that mediates many of its downstream physiological effects. One driving force behind research into DDX3X is its involvement in a range of diseases. For example, DDX3X is an essential host factor used by a diverse range of viruses, such as HIV-1 [bib_ref] Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function, Yedavalli [/bib_ref] [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref] [bib_ref] DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the..., Frohlich [/bib_ref] , HCV [bib_ref] DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication, Ariumi [/bib_ref] [bib_ref] Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to..., Angus [/bib_ref] [bib_ref] Dynamic interaction of stress granules, DDX3X, and IKK-α mediates multiple functions in..., Pène [/bib_ref] and SARS-CoV-2 [bib_ref] Proteomic analysis identifies the RNA helicase DDX3X as a host target against..., Ciccosanti [/bib_ref] [bib_ref] The SARS-CoV-2 RNA-protein interactome in infected human cells, Schmidt [/bib_ref] , making it an attractive target for the development of broad-spectrum antiviral drugs [bib_ref] DEAD-Box RNA helicase DDX3: Functional properties and development of DDX3 inhibitors as..., Kukhanova [/bib_ref] [bib_ref] Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion..., Rao [/bib_ref]. DDX3X has also been found to play a role in several forms of cancer [bib_ref] A doubleedged function of DDX3, as an oncogene or tumor suppressor, in..., He [/bib_ref] , and dysregulation of its role in translation likely mediates at least some of its oncogenic properties. Thus, DDX3X inhibitors are also being explored as anti-cancer drugs (reviewed in [bib_ref] DEAD-Box RNA helicase DDX3: Functional properties and development of DDX3 inhibitors as..., Kukhanova [/bib_ref]. Finally, DDX3X dysfunction has also been linked to neurological diseases. A range of de novo germline DDX3X mutations can cause 'DDX3X syndrome', a neurodevelopmental condition presenting with intellectual disability, microcephaly, and other symptoms, which may be attributed at least in part to altered translational regulation [bib_ref] Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development, Lennox [/bib_ref]. DDX3X has also recently been linked to neurodegenerative conditions caused by translation of neurotoxic peptides [bib_ref] DDX3X and specific initiation factors modulate FMR1 repeatassociated non-AUG-initiated translation, Linsalata [/bib_ref]. With such diverse links to disease, DDX3X is considered a promising drug target. However, it is becoming increasingly apparent that DDX3X's role in homeostatic conditions and disease contexts is complex, and further research to characterise the exact cellular functions of DDX3X is needed. Here, we review the evidence surrounding DDX3X's roles in translation regulation, because this is emerging as one of its key cellular roles, and its dysregulation is likely involved in DDX3Xlinked disease pathophysiology. ## Ddx3x is an rna binding and remodelling protein As a DEAD-box protein, DDX3X's ability to bind RNAs and unwind them is relatively well characterised. However, many questions remain about DDX3X's exact mode of interaction with its RNA targets, and what distinguishes these RNAs from nontarget RNAs. ## Ddx3x's interactions with rna Several biochemical studies have characterised how DDX3X binds its RNA targets. Binding has been observed for both singlestranded (ss) and double-stranded (ds) RNA, however for dsRNA only if there is either a 3′ or a 5′ overhang [bib_ref] HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity, De Bisschop [/bib_ref]. DDX3X binding to RNA is not dependent on the presence of ATP [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X, Song [/bib_ref] and it does not recognise specific bases. Instead, DDX3X interacts mainly with the phospho-ribose backbone which it uses to discriminate RNA from DNA [bib_ref] The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X, Song [/bib_ref]. Thus, DDX3X displays lax binding specificity for RNA substrates in vitro. Yeast Ded1 was shown to be capable of oligomerisation, most commonly as a trimer, wherein two Ded1 molecules bind onto an mRNA to recruit a third molecule which unwinds it. DDX3X has also been found to be capable of oligomerisation [bib_ref] HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity, De Bisschop [/bib_ref] , with a recent crystal structure of DDX3X-RNA complexes showing it as a dimer [bib_ref] The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X, Song [/bib_ref]. DDX3X can unwind RNA-RNA and DNA-RNA duplexes in vitro in the presence of ATP [bib_ref] Biochemical differences and similarities between the DEAD-box helicase orthologs DDX3X and Ded1p, Sharma [/bib_ref] , but not blunt duplexes lacking single-stranded overhangs. It is unclear whether or to what extent DDX3X can unwind DNA-DNA double-strands, with [bib_ref] A motif unique to the human DEAD-box protein DDX3 is important for..., Garbelli [/bib_ref] finding unwinding of 5′ and 3′ DNA-DNA substrates, but [bib_ref] Biochemical differences and similarities between the DEAD-box helicase orthologs DDX3X and Ded1p, Sharma [/bib_ref] finding no appreciable unwinding of such a substate. Based on evidence from X-ray crystallographic structures of both DDX3X and the related D. melanogaster DEAD-box helicase Vasa, [bib_ref] The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X, Song [/bib_ref] suggested a model for DDX3X activity on dsRNA. First, two molecules of DDX3X undergo conformational changes to complex with dsRNA. Next, binding of ATP causes conformational changes resulting in a closed form of the helicase and unwinding of the RNA duplex, probably via bending of the RNA [bib_ref] Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa, Sengoku [/bib_ref]. Finally, the closed form of the helicase permits ATP hydrolysis [bib_ref] Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa, Sengoku [/bib_ref] , which results in release of the unwound RNA. RNA binding stimulates DDX3X's ATP hydrolysis activity, which is often used as a measure of its substrate unwinding activity. de Bisschop et al.found that DDX3X's ATP hydrolysis activity varied depending on the RNA substrate (in the form of truncated HIV-1 genomic RNA (gRNA)) and suggested that RNA structural elements may play a role in stimulating DDX3X's enzymatic activity. In addition to unwinding their RNA substrates, DEAD-box helicases can form long-lived interactions with RNA in an ATPbound state known as "clamping." In the case of the DEAD-box protein eukaryotic initiation factor (eIF) 4A, clamping allows it to act as a nucleator to establish larger protein complexes on mRNAs [bib_ref] From unwinding to clamping -The DEAD box RNA helicase family, Linder [/bib_ref]. Although to date there is not much direct evidence of clamping in the DDX3X/ Ded1 helicase family, their known interactions with a variety of RNA Binding Proteins (RBPs) raises the possibility of them having long-lived interactions with RNA in RBP complexes [bib_ref] A novel Ded1-like RNA helicase interacts with the Y-box protein ctYB-1 in..., Nashchekin [/bib_ref] [bib_ref] The DEAD-box protein Ded1 modulates translation by the formation and resolution of..., Hilliker [/bib_ref]. found that the eIF4A inhibitor Rocaglamide A also binds DDX3X in the cleft between its helicase core and polypurine RNA substrates, which results in a state similar to clamping (although in an ATP-independent manner) and leads to a scanning impediment of the affected mRNAs. Although arising from an exogenous intervention with a small molecule inhibitor, this illustrates the potential for DDX3X to exert inhibitory effects on mRNA translation through long-lived interactions with its targets. ## Ddx3x's binding to physiological rna targets The studies discussed in Section 2.1 defined DDX3X's interactions with RNA using synthetic substrates, but do not address DDX3X's binding to diverse transcripts within the cell. Defining these RNA targets across the transcriptome is important for understanding DDX3X function, as it may exhibit specific binding preferences in cells, which in turn impact downstream effects of translation regulation by DDX3X. To address this, several studies have used Cross-Linking and ImmunoPrecipitation (CLIP) methods coupled with RNAseq (CLIP-seq) to identify DDX3X's cellular RNA targets and its binding sites on a transcriptome-wide scale. These studies mostly agree on the broad patterns of DDX3X RNA binding in the cell. They show that DDX3X binds a large number of transcripts, with one study identifying over 10,000 bound mRNAs. This is not unexpected given that its yeast equivalent Ded1 binds virtually all yeast mRNAs [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref]. Although binding to non-coding RNAs was also observed, DDX3X bound primarily to protein-coding transcripts [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. Binding was observed mainly in exons as opposed to introns, an indication that DDX3X mainly binds mature mRNAs [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. Among the non-coding RNAs, DDX3X has been observed to bind all three rRNAs [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. Although this may be an artefact of the high levels of rRNAs within the cell, DDX3X binding to the 18S rRNA between nucleotides 527 and 553 has been identified as a high-confidence interaction, which may constitute a functionally important direct interaction with the ribosome [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. Across CLIP-seq studies, metagenomic profiling has shown the distribution of DDX3X binding sites across mRNAs. The highest number of crosslinks was observed in the 5′ untranslated region (UTR), which was further enriched at the translation start site [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. DDX3X has also been shown to bind near the transcription start site, which may indicate its loading proximal to the 5′cap through its interactions with cap-binding proteins (see Section 3). Enriched binding of DDX3X to 5′ UTRs suggests a role for DDX3X in translation initiation, as destabilising RNA secondary structures in the 5′UTR may facilitate interactions with the translation machinery and/or contribute to ribosomal scanning, as discussed in Section 3. It is interesting to contrast DDX3X's binding preference for coding mRNA's 5′UTRs observed in CLIP-seq studies with in vitro evidence suggesting that DDX3X binds RNA in a non-specific manner [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X, Song [/bib_ref]. This could suggest that DDX3X binding is influenced by structural features in RNAs or by interactions with other RBPs that were absent from in vitro studies. Of note, CLIP studies have so far not found a clear consensus sequence for DDX3X binding [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. This further supports the idea that DDX3X recognises higher-order structural elements in RNAs rather than sequence motifs; and these might elude traditional motifsearching algorithms. In line with this, [bib_ref] The RNA helicase DDX3 induces neural crest by promoting AKT activity, Perfetto [/bib_ref] showed that translation of both human and Xenopus tropicalis Ras-related C3 botulinum toxin substrate 1 (RAC1) mRNAs, which have poor sequence conservation but similar predicted secondary structure, is dependent on DDX3X. Nevertheless, some CLIP studies have identified broad characteristics of DDX3X RNA binding sites, such as a high GC content and complex secondary structure [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. For example, [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] observed enrichment of predicted G-quadruplex (G4) structures upstream of DDX3X binding sites, as well as GC-rich regions resembling Cysteine-Enriched Regulators of Translation (CERT)-motifs downstream of DDX3X crosslinking sites. As discussed in Section 3, DDX3X's interactions with eIFs may also contribute to the observed binding preferences for 5′UTRs of coding mRNAs. Furthermore, interactions with other RBPs could mediate some of DDX3X's interactions with specific mRNA targets. For example, heterogenous nuclear Ribonucleoprotein K (hnRNPK) was shown to interact with DDX3X and mediate enhanced translation of JUND mRNA during metabolic stress in pancreatic β-cells, possibly through a C-rich motif in the 3′UTR of the JUND mRNA [bib_ref] Metabolic stress activates an ERK/hnRNPK/DDX3X pathway in pancreatic β cells, Good [/bib_ref]. DDX3X was also recently shown to mediate Endoplasmic Reticulum (ER) associated translation of secreted growth factors through interactions with signal recognition particle (SRP) proteins. This was mediated via the 3′UTRs of growth factor mRNAs and did not require DDX3X's helicase activity, thus it seems to constitute a distinct mechanism of DDX3X for regulating translation of mRNAs encoding secreted proteins . Interestingly, both studies suggest that regulation occurs through interactions with 3′UTRs, but open questions remain about the motifs and mechanisms of regulation. Some CLIP-seq studies investigated whether diseaseassociated DDX3X mutations affect its RNA binding.found that DDX3X bearing a medulloblastomaassociated R534H mutation that reduces its catalytic activity had a similar overall binding profile to wild-type DDX3X. Additionally, [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] showed that different medulloblastoma-associated DDX3X mutants (M370R, G302V, G325E) retained their RNA binding ability in vitro. Overall, this suggests that these mutations do not drastically change the binding of DDX3X to its RNA substrates, although they impact downstream consequences of binding (see Section 4.2 and Section 4.3.1). In summary, although some broad characteristics of DDX3X binding patterns have been identified, further investigation into the determinants of DDX3X binding to its physiological RNA targets is needed to better understand its interactions with the transcriptome. Additional biochemical studies to determine which RNA characteristics stimulate DDX3X's ATPase activity would also be useful, as this is likely relevant for its functional interactions with different physiological targets and can influence the downstream consequences of DDX3X-RNA interactions. ## Ddx3x interacts with various components of the translation machinery Many interactions between DDX3X and eIFs, ribosomal proteins, rRNA and other translation-related factors have been described, opening up numerous possibilities for roles in translation regulation. So far, known interactions mainly link DDX3X to translation initiation, in line with its binding enrichment in 5′UTRs and at start codons. However, the mechanistic contributions of DDX3X to these complexes is not fully understood, and the presence of DDX3X in different translation (pre)-initiation complexes may suggest that it can have several distinct or overlapping roles. ## Ddx3x's interactions with cap-binding complexes DDX3X may already be associated with mRNA when it exits the nucleus, as it is a nucleo-cytoplasmic shuttling protein and has been linked to mRNA export [bib_ref] DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the..., Frohlich [/bib_ref] [bib_ref] Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3, Brennan [/bib_ref]. mRNAs that are exported from the nucleus are bound at the 5′cap by the cap-binding complex (CBC) consisting of cap-binding proteins 20 and 80 (CBP-20 and CBP-80) [bib_ref] Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated..., Ishigaki [/bib_ref] , and at the 3′ end by poly-A binding protein (PABP) [bib_ref] Poly(A)-binding proteins and mRNA localization: Who rules the roost?, Gray [/bib_ref]. DDX3X has been found to interact with the CBPs [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref] and PABP, the latter being a direct interaction with the core domain of DDX3X [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref]. Cytosolic mRNAs bound by the CBC are subject to a 'pioneer round' of translation that allows for quality control of new mRNAs and triggering of nonsense-mediated decay (NMD) should a premature stop codon be detected [bib_ref] Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated..., Ishigaki [/bib_ref] [bib_ref] Nonsense-mediated mRNA decay begins where translation ends, Karousis [/bib_ref]. It is possible that DDX3X plays a role in this mechanism, as it has been identified within NMD protein complexes [bib_ref] Identification of interactions in the NMD complex using proximitydependent biotinylation (BioID), Schweingruber [/bib_ref] and plant DDX3X orthologues have been implicated in the NMD process [bib_ref] RNA helicases from the DEA(D/H)-Box family contribute to plant NMD efficiency, Sulkowska [/bib_ref]. It is however also possible that the DDX3X-CBP interactions facilitate a form of alternative translation initiation [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref] , as discussed in Section 4.4.3. Following the pioneer round, CBC proteins are usually exchanged for the cap-binding initiation factor eIF4E, which primes the mRNA for further translation by assembling the eIF4F complex. DDX3X has also been shown to directly bind to eIF4E, with a mapped binding site at a 38-YIPPHLR-44 motif in the DDX3X N-terminus, although the C-terminal region also showed some eIF4E-binding ability [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref]. eIF4E associates with eIF4G and A to form the eIF4F complex, and PABP also binds eIF4G to circularise the mRNA, facilitating efficient translation in polysomes [bib_ref] Protein synthesis initiation in eukaryotic cells, Merrick [/bib_ref]. Numerous experiments have shown an interaction between DDX3X and eIF4G [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref] [bib_ref] Bovine adenovirus-3 pVIII suppresses cap-dependent mRNA translation possibly by interfering with the..., Ayalew [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref] , with direct binding observed between positions 682-1086 of eIF4G [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref]. Direct binding to yeast eIF4G, A and E was also observed with Ded1 [bib_ref] The DEAD-box protein Ded1 modulates translation by the formation and resolution of..., Hilliker [/bib_ref] [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref] , further supporting the notion that DDX3X directly associates with the mRNA-eIF4F complex [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref]. It is also possible that DDX3X can bind directly to the 5′ methylguanosine cap. [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref] found that DDX3X was retained on a 7′mGTP affinity column along with eIF4G and PABP whilst displacing eIF4E, suggesting that it can associate with the 5′cap independent of eIF4E. In line with this, they also found that DDX3X accumulated in cytoplasmic granules containing HIV-1 gRNA, eIF4G, and PABP but not CBP-80 or eIF4E. Based on these results, the authors suggested that DDX3X can substitute for eIF4E in the translation of HIV-1 gRNA. A similar observation was made by [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref] who found that DDX3X favoured recruitment of CBP-20 over eIF4E to the 5′cap of uORF-containing transcripts [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref] , see also Interactions between DDX3X and other RNA-binding proteins during translation (pre)-initiation. (A) Potential DDX3X-containing protein complexes associated with the mRNA 5′cap. Only known DDX3X-interacting factors are included for clarity. (i) DDX3X may be associated with CBC after nuclear export, which could allow it to contribute to pioneer translation and/or nonsense-mediated decay (NMD), or (ii) alternative translation mechanisms independent of eIF4F [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref]. (iii) DDX3X has also been shown to interact with eIF4G [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] and eIF4E, meaning that it might be associated with the conventional eIF4F complex on RNA [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref]. (iv) Alternatively, it may form novel cap-binding complexes with PABP and eIF4G, but independently of eIF4E (Soto-Rifo et al., 2013) (B) Potential contribution of DDX3X helicase activity to recruitment of the 43S PIC to mRNA 5′ends. The presence of stem loops proximal to the 5′ cap can inhibit association of the PIC with the mRNA (left). Through destabilisation of cap-proximal structured regions on the mRNA, DDX3X may support 43S binding (right) [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref]. (C) Potential contribution of DDX3X to scanning. Structured regions within the 5′UTR downstream of the scanning 48S PIC (left) have the potential to stall scanning and possibly terminate translation initiation (Abaeva et al.,. Resolution of these structures by DDX3X may facilitate proper scanning of mRNAs containing long, structured 5′UTRs (right). Frontiers in Cell and Developmental Biology frontiersin.org Section 4.4.3). In contrast, in experiments conducted by [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref] DDX3X was only observed to bind to a 7′mGTP affinity column when eIF4E was also present. In their experiments, DDX3X reduced the level of eIF4G bound to eIF4E in a dose-dependent manner. The authors proposed that DDX3X disrupts the eIF4E-eIF4G interaction and thereby exerts a negative effect on translation initiation, suggesting that DDX3X can also repress translation. It is possible that the conflicting results can be explained by the different experimental set-ups, as Soto-Rifo et al.conducted their analysis using HeLa cell extracts whereas Shih et al.used only purified recombinant proteins and thus other protein co-factors that might modulate the composition of cap-binding complexes were absent in this work. When recruited to the 5′cap, DDX3X can use its helicase activity to destabilise secondary structures that may otherwise interfere with loading of the eIF4F complex or recruitment of the 43S pre-initiation complex (PIC) [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref] and see Section 3.2) [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref] [bib_ref] General and target-specific DExD/H RNA helicases in eukaryotic translation initiation, Shen [/bib_ref]. Further studies are required to determine the exact contributions of DDX3X to different cap-binding protein complexes in vivo. It is possible that DDX3X's roles at the 5′cap differ between viral and host translation, or between mRNAs that undergo conventional versus alternative translation initiation, and as such are influenced by environmental conditions. Future work should further elucidate whether DDX3X is part of alternative capbinding complexes in the absence of eIF4E and/or with CBP-20 and how these complexes function. Intriguingly, some studies suggested modulation of DDX3X/Ded1 recruitment and function in cap-binding complexes by (mammalian) Target of Rapamycin (TOR) [bib_ref] Proteomic analysis of cap-dependent translation identifies LARP1 as a key regulator of..., Tcherkezian [/bib_ref] [bib_ref] Regulation of global and specific mRNA translation by the mTOR signaling pathway, Nandagopal [/bib_ref] [bib_ref] The DEAD-box RNA helicase Ded1 has a role in the translational response..., Aryanpur [/bib_ref] , and Ded1 appears to be required for growth suppression downstream of TOR inhibition [bib_ref] The DEAD-box RNA helicase Ded1 has a role in the translational response..., Aryanpur [/bib_ref] , suggesting that environmental conditions and cellular stress can alter DDX3X's recruitment to and role in cap-binding complexes. ## Ddx3x's interactions with preinitiation complexes The next step in translation pre-initiation involves the association of the 43S pre-initiation complex (consisting of the 40S small ribosomal subunit (SRS), the ternary complex, and eIF3) with the mRNA-eIF4F complex [bib_ref] Protein synthesis initiation in eukaryotic cells, Merrick [/bib_ref]. DDX3X can directly bind to eIF3c [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref] , and has been observed to associate with numerous other eIF3 subunits [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref]. Additionally, DDX3X interacts with a variety of 40S ribosomal components (e.g., RPS5, RPS6, RPS11) [bib_ref] Ribosomal protein L13 promotes IRES-driven translation of foot-and-mouth disease virus in a..., Han [/bib_ref] and there is evidence of direct binding between DDX3X and the 40S ribosome [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref]. In addition to binding ribosomal proteins, DDX3X has been found to bind 18S rRNA at a site mapping to helix 16, which faces incoming mRNA on the small ribosomal subunit . This binding site has also been observed with Ded1 [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref] , suggesting that this is an evolutionarily conserved interaction with the preinitiation complex that allows exposure to mRNAs during the scanning process. The presence of DDX3X within both the mRNA-eIF4F complex and the pre-initiation complex raises numerous possibilities for potential function(s) during translation initiation. As discussed in Section 3.1, it may have a role in the assembly of cap-binding complexes that activate the mRNA for interactions with the 43S pre-initiation complex. Because DDX3X also interacts with components of the 43S PIC, such as eIF3 and the 40S ribosomal subunit, it is also conceivable that DDX3X directly mediates 43S PIC recruitment to (structured) mRNAs. There is some evidence for this in yeast, where Ded1 can promote 48S formation with mRNAs that contain cap-proximal stem loops predicted to interfere with PIC loading. It is conceivable that this mechanism is conserved for DDX3X given that it interacts with eIF4F in a comparable manner to Ded1. However, two studies found no evidence for a contribution of DDX3X to 48S PIC formation. Abaeva et al. (2011) examined formation of 48S complexes with different configurations of eIFs in vitro and found no effect of DDX3X. In another study, knockdown of DDX3X was found to increase rather than decrease the number of 48S complexes [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref] , suggesting that 48S assembly, at least on a global level, can occur in the absence of DDX3X. It is however possible that DDX3X supports 48S assembly only for specific mRNAs, e.g., those containing secondary structures that impede association with the 43S PIC [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref]. An example of this is the HIV-1 gRNA, where DDX3X has been shown to resolve structures at the 5′cap, presumably to enable association with the 43S PIC. This was also observed for several cellular transcripts that contain similar cap-proximal secondary structures, such as NADH Dehydrogenase Ubiquinone 1 alpha subcomplex subunit 1 (NDUFA1) and Interleukin-18 (IL-18) [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref]. It is also a possibility that DDX3X is merely recruited to capbinding complexes but then exerts its real function during the process of scanning [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref]. Scanning of 5′UTRs for a viable start codon takes place after the mRNA-eIF4F and 43S PIC complexes combine. Yeast Ded1 has been implicated in this process; it promotes scanning and AUG recognition in transcripts containing stem loops [fig_ref] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation [/fig_ref] (Abaeva et al., 2011). Ded1 was found to enhance 48S formation on mRNAs containing cap-distal stem loops to an even greater degree than cap-proximal stem loops in an ATPase-dependent manner, suggesting that it supports their destabilisation during the scanning process. Furthermore, profiling of scanning ribosomes showed that deletion of Ded1 caused a shift away from the AUG codon towards the 5′UTR, indicating a reduced rate of scanning [bib_ref] Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex..., Sen [/bib_ref]. Based on CLIP-seq data, [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref] proposed a model for Ded1 function in which it is gradually recruited to the scanning PIC via its Frontiers in Cell and Developmental Biology frontiersin.org interactions with eIFs and rRNA, and resolves secondary structures to accelerate scanning, prevent stalling, and support initiation at the start codon of the protein-coding ORF. In comparison to Ded1, the role of DDX3X in the scanning process is less certain. Soto-Rifo et al.argued against a role of DDX3X in the scanning process in the translation of HIV-1 genomic RNA (gRNA) and other highly structured mRNAs, as insertion of an unstructured sequence upstream of the capproximal stem loop abolished DDX3X dependency, suggesting that DDX3X activity is needed for earlier stages, such as 43S PIC recruitment to the 5′cap complex. On the other hand, knockdown of DDX3X resulted in a global shift in ribosome occupancy from the coding sequence towards the 5′UTR , suggesting a defect in scanning rather than 43S PIC recruitment or other earlier stages of pre-initiation. It is also interesting that medulloblastoma-associated DDX3X mutations caused scanning defects when introduced into Ded1,, indicating a possible conservation in function between the orthologues. ## Ddx3x's interactions with the 80s ribosome Upon finding a start codon in good sequence context, remodelling of the 48S complex takes place and the 60S large ribosomal subunit joins to form the complete 80S ribosome and initiate translation. DDX3X was found to bind the 60S large ribosomal subunit (LRS), mediated through a direct interaction with ribosomal protein L13 (RPL13) [bib_ref] Ribosomal protein L13 promotes IRES-driven translation of foot-and-mouth disease virus in a..., Han [/bib_ref]. It also binds to 5.8S rRNA and 28S rRNA, although this might be a less significant interaction than with 18S rRNA [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref]. It is possible that DDX3X assists in ribosome assembly through these protein-protein and protein-RNA interactions, as one study found it to be associated with newly formed 80S ribosomes, and loss of DDX3X decreased the number of newly formed ribosomes [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref]. ## Involvement of ddx3x in translation elongation and ribosome recycling To date, most studies have examined DDX3X's role in translation initiation and not much is known about DDX3X involvement in other stages of translation. Polysome profiling experiments in mammalian cells found that DDX3X associates mainly with pre-initiation complexes and newly formed 80S ribosomes but is not detected in heavier fractions that represent polysomes engaged in translation elongation [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref]. Nevertheless, [bib_ref] Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of..., Padmanabhan [/bib_ref] have detected Leishmania DDX3 in the polysome fraction and identified perturbations in elongation and ribosome recycling after DDX3 knockdown. CLIP-seq studies have also consistently identified DDX3X binding to both CDSs and 3′UTRs, which may indicate a (transient) association with translating ribosomes, however this is much less pronounced than binding in 5′UTRs (see Section 2.2). Further studies would be required to examine whether roles in elongation and ribosome recycling are conserved in mammalian DDX3X. In summary, through numerous interactions with components of the translation machinery, DDX3X likely maintains an association with the mRNA throughout translation pre-initiation, initiation, and possibly beyond. This gives it opportunities to regulate translation at a variety of stages, including 5′cap recognition and mRNA activation, 43S PIC recruitment, scanning, and ribosomal subunit joining. Although further detailed analysis is needed, it can be speculated that DDX3X may exert several distinct, possibly independent functions at multiple stages of translation initiation, the exact nature of which may depend on structural features in the specific mRNA transcript and the cellular environment. ## Translation regulation by ddx3x and its physiological consequences Through the aforementioned CLIP-seq experiments, a growing number of mRNAs are being defined as DDX3X targets in various contexts, such as neurodevelopment and cancer However, for many of the mRNAs that were identified in CLIP studies as DDX3X targets, we have no follow-on studies that analyse their regulation by DDX3X. In this section, we will discuss some of DDX3X's better described mRNA targets and the (potential) physiological consequences of their regulation. ## Examples of ddx3x mrna targets Across the available studies, several mRNAs have repeatedly been identified as DDX3X targets, and some studies have investigated mechanisms and downstream physiological consequences of DDX3X-mediated translation regulation of specific mRNAs (for examples of DDX3X targets, see [fig_ref] TABLE 1: Examples of DDX3X targets in the literature [/fig_ref]. Cyclin E1 is one of the best studied DDX3X targets, and its 5′UTR has been used as a positive control for assessing DDX3Xmediated translation regulation in subsequent studies [bib_ref] DDX3 functions in antiviral innate immunity through translational control of PACT, Lai [/bib_ref]. Regulation of Cyclin E translation linked DDX3X to cell growth control and G1/S regulation, which carries strong implications for its role in cancer [bib_ref] DDX3 regulates cell growth through translational control of cyclin E1, Lai [/bib_ref]. Another recurring DDX3X target is Ornithine Decarboxylase 1 (ODC1), which possesses a long, structured 5′UTR that inhibits its translation [bib_ref] The translation in vitro of rat ornithine decarboxylase mRNA is blocked by..., Van Steeg [/bib_ref]. RAC1 is another DDX3X target documented across numerous studies, regulation of which may contribute to DDX3X's roles in neurological development [bib_ref] The RNA helicase DDX3 induces neural crest by promoting AKT activity, Perfetto [/bib_ref] , cancer cell metastasis [bib_ref] DDX3 modulates cell adhesion and motility and cancer cell metastasis via Rac1-mediated..., Chen [/bib_ref] , and macrophage phagocytosis . Several other Frontiers in Cell and Developmental Biology frontiersin.org (2019) Calviello et al. Perfetto et al. IL-18 LUC HeLa Immunity Soto-Rifo et al. IL-8 LUC HeLa Immunity Soto-Rifo et al. Line-1 LUC HeLa -Soto-Rifo et al. Lai et al. (2013) Soto-Rifo et al. de Bisschop et al. Frohlich et al. Frontiers in Cell and Developmental Biology frontiersin.org DDX3X targets have been linked to cancer, implicating DDX3X in tumorigenesis. For example, DDX3X has been found to regulate both cap-and IRES-dependent translation of the prominent tumour suppressor p53. In melanoma cells, DDX3X-mediated regulation of Microphthalmia-associated transcription factor (MITF) translation was shown to be important for the metastatic phenotype [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref]. In prostate cancer cells, androgen receptor (AR) translation is regulated by DDX3, which impacts castration-resistance in this cancer type [bib_ref] RNAbinding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of..., Vellky [/bib_ref]. Interestingly, it is negatively regulated by DDX3, likely via sequestration of AR mRNA within stress granules (see Section 4.3.1). Amphiregulin (AREG) is a secreted growth factor whose translation is also regulated by DDX3X, in concert with SRP, to promote translation at the ER in the secretory protein synthesis pathway (see also Section 2.2). In addition to RAC1, several other DDX3X targets are involved in neurodevelopment. The 5′UTR of Protein Kinase A (PKA) is similar to that of RAC1, and its translation is also regulated by DDX3X, affecting neurite development [bib_ref] DDX3 modulates neurite development via translationally activating an RNA regulon involved in..., Chen [/bib_ref]. Through ribosome profiling in a murine cortical development model,identified a host of DDX3X-regulated genes involved in neurological development, such as REST Corepressor 2 (Rcor2), SET Domain-Containing Protein 3 (Setd3) and DNA Topoisomerase II Binding Protein 1 (Topbp1). These multiple links to translation of mRNAs for neurodevelopmental genes may provide insights into the causes of DDX3X-associated intellectual disability (DDX3X syndrome). DDX3X also translationally regulates a suite of immune genes, including Transforming Growth Factor ß (TGF-ß) [bib_ref] The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well..., Lai [/bib_ref] , IL-8, , signal transducer and activator of translation 1 (STAT1), Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1), and Protein Activator of Interferon Induced Protein Kinase EIF2AK2 (PACT) . The effect on PACT allows DDX3X to indirectly regulate anti-viral innate immune responses, such as interferon ß (IFN-ß) production [bib_ref] DDX3 functions in antiviral innate immunity through translational control of PACT, Lai [/bib_ref]. DDX3X also regulates translation of mRNAs encoding mitochondrial proteins, such as Cytochrome C Oxidase II (MT-COII) and NADH Dehydrogenase 1 (MT-NDI), linking DDX3X to energy metabolism [bib_ref] Targeting mitochondrial translation by inhibiting DDX3: A novel radiosensitization strategy for cancer..., Heerma Van Voss [/bib_ref]. Finally, another important class of DDX3X targets encode proteins involved in translation [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref] , including large and small ribosomal subunit proteins [bib_ref] RNA G-quadruplex structures control ribosomal protein production, Varshney [/bib_ref] , suggesting that DDX3X may indirectly influence translation more broadly (see also Section 4.2). ## Does ddx3x contribute to global mrna translation? The overall scope of DDX3X's involvement in mRNA translation is still somewhat controversial, despite numerous studies that have interrogated this aspect of DDX3X function. Some reports characterised it as a general regulator of translation, while others maintained that it is only required for translation of particular mRNA subsets, such as those with highly structured 5′UTRs. This discrepancy is seen across several types of experimental evidence. For example, several studies have analysed effects of DDX3X knockdown on polysome profiles within cells. Most of these studies found that DDX3X knockdown did not affect the overall proportions of polysomes, monosomes, 40S or 60S ribosomal subunits [bib_ref] The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well..., Lai [/bib_ref] [bib_ref] DDX3X and specific initiation factors modulate FMR1 repeatassociated non-AUG-initiated translation, Linsalata [/bib_ref] , which indicates that global ribosome occupancy and relative rates of initiation/elongation/recycling are not impacted by DDX3X. However, in some studies DDX3X knockdown did reduce the overall proportion of polysomes, suggesting a global defect in translation [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref] [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref]. This is similar to yeast Ded1, where mutations that confer temperature-sensitivity [bib_ref] Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast..., Sen [/bib_ref] and abrogate Ded1-eIF4A/E interactionswere found to reduce the polysome/monosome ratio, suggesting a broad role for Ded1 in regulating translation. Conflicting reports also come from studies using 5′UTR luciferase reporter assays as read-outs of DDX3X-mediated translation regulation. Several studies found that DDX3X selectively regulated transcripts with highly structured 5′UTRs and did not affect translation efficiency of reporter mRNAs containing various unstructured 5′UTRs [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and..., Çelik [/bib_ref]. However, Geissler et al.observed negative effects on translation of both structured and unstructured 5′UTR mRNA reporters upon DDX3X knockdown. Additionally, studies that assessed global protein synthesis using labelled methionine incorporation are also in disagreement, with some showing no effect of DDX3X knockdown on global translation [bib_ref] DDX3 regulates cell growth through translational control of cyclin E1, Lai [/bib_ref] [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] , whereas others showed decreases [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref] [bib_ref] Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of..., Padmanabhan [/bib_ref] , or even increases [bib_ref] Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting..., Shih [/bib_ref]. Several studies have used ribosome or polysome profiling to characterise transcriptome-wide changes in ribosome/polysome occupancy upon DDX3X or Ded1 manipulation. In yeast, mutations in Ded1 that confer heat or cold sensitivity repressed translation globally but had more pronounced effects on a select number of mRNAs with long, structured 5′UTRs, consequently dubbed Ded1 hyper-dependent mRNAs [bib_ref] Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast..., Sen [/bib_ref] [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref]. Therefore, although Ded1 is a general translation factor in yeast, it also specifically supports translation of a distinct subset of mRNAs. For DDX3X, many studies altered ribosome occupancy only in particular mRNAs, consequently characterised as specific DDX3X targets. Interestingly, generally only a few hundred mRNAs showed changes in ribosome occupancy upon DDX3X manipulation, much fewer than the many 1000s of mRNAs bound by DDX3X in CLIP experiments [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. Changes in Frontiers in Cell and Developmental Biology frontiersin.org translation efficiency of these specific transcripts were observed with DDX3X silencing [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref] , conditional knockout, induced DDX3X degradation , and expression of disease-associated DDX3X mutants [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. In most studies, inactivation of DDX3X increased translation efficiency of only a few mRNAs, while most targets had decreased translation efficiencies [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. In contrast, a recent integrated analysis of 80 human heart translatomes found translation efficiency of most DDX3X targets negatively correlated with DDX3X expression levels [bib_ref] The SARS-CoV-2 RNA-protein interactome in infected human cells, Schmidt [/bib_ref]. In addition, higher predicted structural stability of the target mRNA's 5′UTR was observed in negatively correlating translation efficiency targets [bib_ref] The SARS-CoV-2 RNA-protein interactome in infected human cells, Schmidt [/bib_ref] , contradicting findings from many other studies where DDX3X positively regulated translation of highly structured mRNAs. Of note, this is the only study where DDX3X levels were not manipulated experimentally. Instead, endogenous levels of DDX3X were correlated with translation efficiency of its targets. This may suggest that increased endogenous DDX3X expression can have suppressive effects on target translation; or it could be a tissuespecific difference in DDX3X function. In addition to effects on specific DDX3X target mRNAs, some studies noted global shifts in ribosome occupancy from the CDS to the 5′UTR upon DDX3X knockdown, possibly related to defects in scanning [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] DDX3 depletion represses translation of mRNAs with complex 5' UTRs, Calviello [/bib_ref] and indicating that DDX3X can affect translation more globally. We propose two likely explanations for the occasionally observed broad effects of DDX3X manipulation on translation. Firstly, it has emerged in recent years that mRNAs encoding ribosomal proteins are among DDX3X's specific targets. In CLIP and Ribo-seq studies, gene ontology analyses showed enrichment of terms relating to the translation machinery [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref]. This was also observed in proteomic analyses of DDX3X-depleted cells, suggesting that DDX3X knockdown does indeed reduce expression levels of many ribosomal proteins . Two recent studies provided a mechanism for this by demonstrating that DDX3X binds to G-quadruplex regions present in 5′UTRs of mRNAs for several ribosomal proteins; and that this regulates their translation and affects their overall protein levels [bib_ref] Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel..., Herdy [/bib_ref] [bib_ref] RNA G-quadruplex structures control ribosomal protein production, Varshney [/bib_ref]. It is therefore conceivable that DDX3X inactivation indirectly affects translation more broadly through regulation of ribosomal protein expression. This can explain conflicting findings in knockdown studies, as transient and short-term DDX3X knockdown would only reveal direct effects on specific DDX3X mRNA targets, whereas sustained silencing may cause indirect effects on ribosome biogenesis that eventually impact global translation. In support of this hypothesis, [bib_ref] Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven..., Gong [/bib_ref] found a decrease in global protein synthesis 48 h but not 24 h post-DDX3 silencing. The second explanation for general effects of DDX3X manipulation on translation relates to its role in stress granule formation discussed in Section 4.3.1. Translation of mRNAs that are sequestered in stress granules is suppressed and it is likely that DDX3X plays a role in shuttling mRNAs into and possibly out of stress granules. As discussed before, DDX3X does associate with a substantial proportion of the protein-coding transcriptome in CLIP-seq studies, but under homeostatic conditions only regulates translation efficiency of a minority of the mRNAs it binds to. Under cellular stress conditions however, DDX3X's broad mRNA-binding capacity might allow it to sequester a diverse pool of transcripts into stress granules and inhibit their cap-dependent translation. Indeed, the formation of stress granules has been shown to explain the broad suppression of translation observed upon expression of certain medulloblastoma-associated DDX3X mutants, such as G325E, which have reduced enzymatic activity and trigger aberrant stress granule formation in cells [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref]. The suppressive effect of DDX3X in this case even extends to nontargets that are sequestered into the DDX3X-induced stress granules by other RBPs (see also Section 4.3.1) Stress granulemediated effects are most likely to impact studies using ectopic overexpression of DDX3X and/or analysing effects of inactivating DDX3X mutations. ## Ddx3x regulates translational reprogramming during cellular stress Stress-induced translational reprogramming allows cells to rapidly respond to altered environmental conditions and to conserve energy by restricting translation to mRNAs necessary for survival and defence [bib_ref] Translational control under stress: Reshaping the translatome, Advani [/bib_ref]. At the molecular level, two stress response pathways affect availability of key translation initiation factors. The Integrated Stress Response (ISR) leads to phosphorylation of eIF2α by stress-responsive kinases. This prevents 'recycling' of eIF2 during scanning of 5′UTRs and thereby stalls PICs and induces formation of stress granules [bib_ref] Translational control under stress: Reshaping the translatome, Advani [/bib_ref]. The second pathway is mediated by mTOR and limits availability of eIF4E through eIF4E-binding proteins [bib_ref] mTOR signaling in growth control and disease, Laplante [/bib_ref]. Due to reduced availability of eIF4E and eIF2 during cellular stress, canonical cap-and scanning-dependent translation initiation is suppressed; and mRNAs that are actively translated during these conditions likely rely on noncanonical translation initiation mechanisms. Non-canonical translation initiation mechanisms are diverse and can be mediated for example by Internal Ribosome Entry Sites (IRES), upstream open reading frames (uORFs), capindependent translation elements (CITEs), G-quadruplexes, or epigenetic modifications to mRNAs (reviewed in Frontiers in Cell and Developmental Biology frontiersin.org [bib_ref] Noncanonical translation initiation in eukaryotes, Kwan [/bib_ref]. Much remains to be learned about the exact molecular mechanisms that mediate translation initiation from these different elements, even with respect to their requirements for canonical initiation factors. However, there is increasing evidence that DDX3X is specifically involved in non-canonical translation initiation during cellular stress, which likely contributes to the altered translational responses to stress observed with medulloblastoma-associated DDX3X mutants [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref]. ## Ddx3x's role in stress granule formation and function As mentioned before, regulation of stress granule (SG) formation is another significant process by which DDX3X may affect translation, both globally and for specific target mRNAs. SGs are dynamic liquid-liquid phase separated accumulations of stalled pre-initiation complexes, mRNAs, and various RNA binding-proteins that form in response to cellular stress. Their formation is triggered by disruption of translation and collapse of cellular polysomes, often but not always downstream of eIF2α phosphorylation by stress-sensing kinases (reviewed in [bib_ref] Molecular mechanisms of stress granule assembly and disassembly, Hofmann [/bib_ref]. SG formation drastically reshapes cellular translation through the sequestering of mRNAs, RBPs, and core translation machinery components. In addition, SGs can also sequester signalling proteins and thereby regulate signalling pathways linked to cell survival and immune defence [bib_ref] Stress granules and cell signaling: More than just a passing phase?, Kedersha [/bib_ref]. When the stress is removed or cells adapt to the stress, disassembly of SGs and resumption of translation can occur. Various protein-protein, RNA-protein, and RNA-RNA interactions are involved in mediating liquid-liquid phase separation of SG components [bib_ref] Molecular mechanisms of stress granule assembly and disassembly, Hofmann [/bib_ref]. Given that it binds many different mRNAs and translation machinery components (see section 2 and Section 3), it is unsurprising that DDX3X is detectable in SGs in response to a variety of stresses [bib_ref] The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well..., Lai [/bib_ref] [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref] [bib_ref] Bovine adenovirus-3 pVIII suppresses cap-dependent mRNA translation possibly by interfering with the..., Ayalew [/bib_ref] [bib_ref] DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression, Adjibade [/bib_ref]. DDX3X incorporation into SGs has been suggested to be dependent on its interaction with eIF4E (see Section 3.1) [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref]. In line with this, recruitment to SGs was shown to be prevented by loss of DDX3X's N-terminal low complexity domain (amino acid positions 1-115) [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] , which may be due to an abrogated interaction with eIF4E and/or a reduced propensity for liquid-liquid phase separation [bib_ref] Acetylation of intrinsically disordered regions regulates phase separation, Saito [/bib_ref]. The N-terminus of DDX3X (aa 1-100) was capable of SG induction when ectopically expressed in cells [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref] , further supporting the importance of this region for SG recruitment/formation. [bib_ref] Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly, Cui [/bib_ref] found that treatment with the DDX3X inhibitor RK-33 impaired arseniteinduced SG assembly and suggested that this is due to decreased RNA binding upon loss of DDX3X's ATP-binding. It is therefore possible that both protein-protein and protein-RNA interactions mediate DDX3X's involvement in SG formation. In a similar fashion to many other SG proteins, overexpression of DDX3X can trigger spontaneous SG formation [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref]. Several diseaseassociated DDX3X mutations are more prone to triggering spontaneous SGs than wild-type DDX3X, and it is thought that this contributes to disease pathophysiology in medulloblastoma and neurodevelopmental delay (DDX3X syndrome) [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development, Lennox [/bib_ref]. Vice versa, depletion of DDX3X has been reported to prevent SG assembly [bib_ref] Critical roles of RNA helicase DDX3 and its interactions with eIF4E/ PABP1..., Shih [/bib_ref] [bib_ref] Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly, Cui [/bib_ref] , but not in all cases [bib_ref] The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well..., Lai [/bib_ref] [bib_ref] DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression, Adjibade [/bib_ref] , a discrepancy which may be due to DDX3X knockdown efficiency but could also point to a non-essential function for DDX3X in SG formation. More recent studies suggest that DDX3X might have a role in SG maturation but not their initial formation [bib_ref] ATPase-modulated stress granules contain a diverse proteome and substructure, Jain [/bib_ref] [bib_ref] Acetylation of intrinsically disordered regions regulates phase separation, Saito [/bib_ref]. In being recruited to SGs, DDX3X also sequesters its bound mRNAs, which likely constitutes a form of translational repression by DDX3X [bib_ref] Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation, Valentin-Vega [/bib_ref] [bib_ref] RNAbinding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of..., Vellky [/bib_ref]. As discussed in Section 4.2, DDX3X mutants that trigger spontaneous SG formation induced translational silencing of DDX3X targets and non-targets that was dependent on SGs. Thus, it is likely that DDX3X acts as a broad repressor of canonical translation through its involvement in SG formation under cellular stress conditions and/or when DDX3X's enzymatic activity is impaired by mutation or other interventions. DDX3X can also repress translation of specific target mRNAs by sequestering them in SGs as has been shown for androgen receptor (AR) mRNA [bib_ref] RNAbinding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of..., Vellky [/bib_ref]. An interesting question is whether DDX3X can shuttle specific target mRNAs not only into but also out of SGs. The enzymatic cycle of DEAD-box helicases certainly allows them to dynamically modulate its interactions with target mRNAs, e.g., to release sequestered RNAs upon stimulation of its ATP hydrolysis activity. This has been proposed for Ded1 [bib_ref] The DEAD-box protein Ded1 modulates translation by the formation and resolution of..., Hilliker [/bib_ref] , which was implicated in regulating both the initial response to cellular stress and the recovery phase where translation is resumed [bib_ref] The DEAD-box RNA helicase Ded1 has a role in the translational response..., Aryanpur [/bib_ref]. Similarly, found that expression of DDX3X ameliorated aberrant SG dynamics associated with knockdown of Gle1, a protein that can stimulate DDX3X's ATPase activity. They proposed that DDX3X may interact with Gle1 to aid both assembly and disassembly of SGs in response to heat shock. [bib_ref] Acetylation of intrinsically disordered regions regulates phase separation, Saito [/bib_ref] showed that posttranslational modifications in the N-terminal intrinsically disordered region (IDR) of DDX3X can regulate its incorporation into SGs. They found that lysine residues within this region are acetylated under homeostatic conditions, which prevents liquid-liquid phase separation. Upon cellular stress, DDX3X was recruited to SGs, where it associated with HDAC6 that deacetylated the lysine residues in DDX3X's IDR and thereby enhanced its SG association. Deacetylation of DDX3X contributed to SG Frontiers in Cell and Developmental Biology frontiersin.org maturation but not initiation. These examples demonstrate that DDX3X's function in SGs can be modulated by cellular interaction partners, likely affecting its role in SG dynamics and translational reprogramming. It is also an interesting question whether all DDX3Xcontaining granules that were observed in cells are translationally inactive SGs. SGs are notoriously difficult to biochemically characterise, and most studies so far relied on immunofluorescent staining of core SG components. It is likely that there is a diversity of LLP-separated RNP granules with different compositions and functions dependent on the exact cellular conditions. Intriguingly, it was recently discovered that liquid-liquid phase separation of RBPs can also promote translation of bound mRNAs. The RBP Fragile X Mental Retardation Syndrome-Related Protein 1 (FXR1) was shown to actively recruit and promote translation of its target mRNAs after undergoing LLPS [bib_ref] LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs, Kang [/bib_ref]. It is conceivable that DDX3X may possess a similar function. Previously, puromycin-staining of cells expressing R475G mutant DDX3X revealed the presence of DDX3X in granules that co-localised with puromycin-labelled nascent peptide chains, suggesting active translation or stalled polysomes in or near these SG-like bodies [bib_ref] Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development, Lennox [/bib_ref]. It is also worth considering how DDX3X's recruitment into SGs affects its involvement in cell signalling pathways. DDX3X has a positive role in NLRP3 inflammasome activation, thus promoting release of the pro-inflammatory cytokine IL-1β and inflammatory cell death through pyroptosis, however, its recruitment into SGs during cellular stress prevents this [bib_ref] DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3..., Samir [/bib_ref]. Thus, DDX3X's recruitment into SGs contributes to cell survival under conditions that can trigger SG formation and NLRP3 inflammasome activation. Previously, it had been shown that recruitment into SGs facilitates an interaction between DDX3X and I kappa B kinase alpha (IKKα) during HCV infection, leading to IKKα activation and downstream upregulation of lipogenesis [bib_ref] Dynamic interaction of stress granules, DDX3X, and IKK-α mediates multiple functions in..., Pène [/bib_ref]. It is currently unknown whether SG localisation also plays a role in DDX3X's function in the anti-viral RIG-I signalling pathway. However, it is possible that a link exists because RIG-I has been shown to be recruited into so-called anti-viral SGs that promote RNA ligand-binding and downstream signalling . Overall, DDX3X has many links to stress granule dynamics and the integrated stress response, which may overlap with its role as a regulator of translation. ## Ddx3 as a mediator of non-canonical translation initiation During conditions of cellular stress, the translatome is remodelled significantly and non-canonical translation initiation mechanisms become more prevalent, allowing translation of specific mRNAs while canonical cap-and scanning-dependent translation is blocked. DDX3X has been implicated in regulating various forms of non-canonical translation initiation, occurring in different physiological contexts. ## Ddx3x's interaction with internal ribosome entry sites (ires) As indicated by their name, Internal Ribosome Entry Sites (IRES) mediate direct ribosome recruitment to internal positions in mRNA transcripts and thereby facilitate translation initiation in a cap-independent and, in most cases, scanning-independent manner [bib_ref] IRES-mediated cap-independent translation, a path leading to hidden proteome, Yang [/bib_ref]. IRES are common in viral RNAs where they form highly conserved secondary structures and can be categorised into four classes: class I is able to initiate translation without the need for protein co-factors or initiator tRNA while classes II-IV have an increasing requirement for canonical translation initiation factors and/or additional proteins called IRES-transacting factors (ITAFs) (reviewed in [bib_ref] IRES-mediated cap-independent translation, a path leading to hidden proteome, Yang [/bib_ref]. DDX3X has been shown to bind to and support translation initiation from several viral IRES, as discussed further in Section 5. In short, DDX3X promotes translation from IRES contained in 5′UTRs of many positivesense single-stranded RNA viruses, explaining its role as pro-viral host factor for these viruses. For cellular IRES, DDX3X's role is less clear, possibly because most cellular IRES-containing transcripts can also be translated in a conventional cap-dependent manner, and because they are not as well defined as viral IRES. Up to 15% of human genes were suggested to contain IRES and to be translated in an IRES-dependent manner during cellular stress (reviewed in [bib_ref] Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to..., Leppek [/bib_ref]. Some evidence links DDX3X to regulation of such transcripts, where it could conceivably act as an ITAF that mediates eIF and ribosome recruitment or remodels RNA structures, something that is required for many cellular IRES (reviewed in [bib_ref] Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to..., Leppek [/bib_ref]. [bib_ref] The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Phung [/bib_ref] identified an IRES in the human MITF-M mRNA as a regulatory target of DDX3X when analysing effects of DDX3X knockdown on translation in melanoma cell lines. Translation of this transcript was strongly suppressed in DDX3X knockdown cells, and the authors demonstrated that DDX3X mediates translation of MITF-M in a cap-independent manner via a structural element in its 5′UTR that served as an IRES. In their study, DDX3X knockdown also strongly affected translation from the HCV IRES, but not from other cellular IRES, such as those in the p27 and BCL-XL 5′UTRs. This suggests an interesting specificity for DDX3X's regulation of different IRES, and further research should be conducted to identify the cis and/or trans factors that influence DDX3X's differential effects on specific viral and cellular IRES. Of note, upstream ORFs and G-quadruplex regions have also been shown to influence cellular IRES activity (reviewed in [bib_ref] Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to..., Leppek [/bib_ref] , thus effects of DDX3X on these elements (discussed in Section 4.4.2 and Section 4.4.3) could be interlinked with its potential ITAF function. Frontiers in Cell and Developmental Biology frontiersin.org 4.4.2 DDX3 can remodel G-quadruplex regions in mRNA G-quadruplexes (G4s) are higher-order, stable structures that can be formed by guanine-rich nucleotide sequences in both DNA and RNA. G4s in mRNAs can influence translation efficiency, e.g., in 5′UTRs they impede recruitment of the 43 PIC or ribosome scanning and thereby suppress canonical cap-dependent translation (reviewed in [bib_ref] Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to..., Leppek [/bib_ref]. G4s can also influence cap-independent translation initiation, exerting either positive or negative effects depending on the specific transcript. For example, it has been shown that G4 structures can act as IRES elements, thus facilitating alternative cap-independent translation of these transcripts (reviewed in [bib_ref] Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to..., Leppek [/bib_ref]. On the other hand, there are also examples where G4 elements have negative regulatory effects on IRES-mediated translation. G4binding proteins have been identified, including several DExD/ H-box helicases that can unwind G4 structures, such as DHX36, DDX21, and DHX9 (reviewed in [bib_ref] Action and function of helicases on RNA G-quadruplexes, Caterino [/bib_ref]. When [bib_ref] Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel..., Herdy [/bib_ref] conducted an unbiased screen for proteins that bind the G4 element in NRAS mRNA, they identified DDX3X as an additional G4-binding protein. They then identified a wide range of DDX3X binding sites primarily in 5′UTRs that correspond to G4 motifs. They demonstrated that DDX3X interacts with G4s via its Glycine-Arginine-rich (GAR) region. In a follow-on study, the authors demonstrated that G4regions are prevalent in mRNAs encoding ribosomal proteins and that knockdown of DDX3X can suppress synthesis of the encoded ribosomal proteins [bib_ref] RNA G-quadruplex structures control ribosomal protein production, Varshney [/bib_ref]. Similar findings were also published for Ded1, with these studies additionally showing that Ded1 binding destabilises G4s in an ATP-independent manner [bib_ref] DEAD-box RNA helicases Dbp2, Ded1 and Mss116 bind to G-quadruplex nucleic acids..., Gao [/bib_ref] [bib_ref] The RGG domain in the C-terminus of the DEAD box helicases Dbp2..., Yan [/bib_ref]. Regulation of G4-containing transcripts by DDX3X should be a focus of further research, as other studies also identified enriched binding of DDX3X to G-rich regions, and G4s might be linked to DDX3X's differential effects on . ## Ddx3x's role in upstream orf (uorf) translation and initiation site selection For eukaryotic mRNAs, translation initiation usually begins at the first AUG start codon in the transcript, mediated by ribosomal scanning that starts at the 5′cap. However, it is now known that significant numbers of translation initiation events do not adhere to this rule. Successful initiation depends on the sequence context around the AUG, with strong AUG start codons embedded into a consensus Kozak sequence [bib_ref] Point mutations define a sequence flanking the AUG initiator codon that modulates..., Kozak [/bib_ref]. AUGs with weaker sequence context can be skipped during ribosomal scanning. Alternative start codon usage can lead to expression of N-terminally truncated or extended protein isoforms compared to the predominant isoform. In many cases however, upstream AUGs do not lead to expression of alternative protein isoforms, but are part of so-called upstream open reading frames (uORFs). uORFs are short open reading frames found upstream of the main protein-coding ORF, sometimes overlapping with it. These are surprisingly prevalent in the human genome; it is now thought that at least 50% of human genes have upstream ORFs [bib_ref] Non-AUG translation initiation in mammals, Andreev [/bib_ref]. While some uORFs express small functional peptides, uORFs are mainly thought to regulate translation efficiency of the protein-coding ORF by preventing re-initiation at the main AUG. Due to this mechanism, most uORF-containing mRNAs have low constitutive translation levels. During cellular stress, when eIF2 levels are limited, ribosomes are more likely to skip the uORF and initiate at the main AUG start codon, leading to increased expression of the encoded protein. The standard example for this type of regulation is Activating Transcription Factor (ATF) 4, a key regulator of the integrated stress response. The ATF4 mRNA contains three uORFs, one of which is overlapping with the main protein-coding ORF. During cellular stress, ATF4 translation is enhanced as initiation occurs more frequently at the AUG for the protein-coding ORF. [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref] demonstrated that DDX3X favours translation of the main ORF in the ATF4 transcript and other select uORF-containing transcripts (ATF5 and DDIT3). Interestingly however, not all uORF-containing transcripts were equally regulated by DDX3X, as CCAAT/Enhancer-Binding Protein Alpha (CEBPA) and ETS Translocation Variant 1 (ETV1) uORF-containing transcripts were unaffected by DDX3X depletion in this study. The authors suggested that DDX3X primarily affects translation initiation in long uORF-containing transcripts, as extension of the short uORFs in CEBPA and ETV1 rendered these transcripts more dependent on DDX3X. The study also demonstrated that presence of DDX3X favoured recruitment of CBP20 rather than eIF4e to the 5′cap of these transcripts, suggesting a possible involvement of the CBC in regulating alternative translation initiation in these uORF-containing transcripts (see also Section 3.1). [bib_ref] DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression, Adjibade [/bib_ref] had also previously demonstrated a positive role for DDX3X in regulating translation of the ATF4 transcript. In contrast to [bib_ref] DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in..., Chen [/bib_ref] , these authors suggested that DDX3X associates with the classical eIF4F complex (including eIF4E) and regulates translation of the ATF4 transcript through this. Initiation in uORFs can occur at standard AUG codons, but also at near-cognate codons, such as CUG, the most efficient non-AUG initiation codon [bib_ref] Non-AUG translation initiation in mammals, Andreev [/bib_ref]. DDX3X's yeast homologue Ded1 was shown to prevent initiation at non-AUG start codons that are immediately upstream of RNA secondary structures. It was suggested that Ded1 accelerates unwinding of these secondary structures and thereby reduces the dwell time of the ribosome. This prevents initiation at the structure-adjacent non-AUG codons, favouring translation of the protein-coding ORF [bib_ref] The helicase Ded1p controls use of near-cognate translation initiation codons in 5'..., Guenther [/bib_ref]. Human DDX3X has also been implicated in regulating initiation from non-AUG codons in the context of repeat-associated non-AUG translation (see Section Frontiers in Cell and Developmental Biology frontiersin.org 4.4.4). In addition, DDX3X's propensity to suppress translation of non-AUG uORFs was recently suggested to play a role for DDX3X's contribution to translation of HIV-1 transcripts. Several HIV-1 transcripts contain non-AUG uORFs that reduce translation of the main proteincoding ORF. Transcripts with non-AUG uORFs were particularly sensitive to DDX3X depletion, suggesting that DDX3X prevents translation initiation at the suppressive uORFs to favour translation of the main protein-coding ORF, very much like what has been proposed for Ded1. This may well be relevant for DDX3X's function as an essential host factor for HIV-1 replication. Interestingly, the study also demonstrated that uORF-encoded peptides stimulated specific T cell responses in HIV-1 infected samples, suggesting that blocking DDX3X activity in HIV-1 infection could suppress viral protein expression and replication while simultaneously enhancing production of antigenic peptides derived from uORFs. Regulation of translation efficiency in uORF-containing transcripts is influenced by multiple factors, such as sequence context of the initiation codons and adjacent structural elements, spacing and lengths of uORFs, epigenetic modifications of the mRNA (m6A), and transacting factors, such as eIF3 subunits, eIF1 and eIF5, and cap-binding complex (CBC) (reviewed in [bib_ref] uORF-mediated translational control: recently elucidated mechanisms and implications in cancer, Chen [/bib_ref]. While structural elements likely play a role, more work is required to elucidate cis and/or trans factors that render a uORF-containing transcript sensitive to DDX3X regulation. ## Ddx3x regulates repeat-associated non-aug (ran) translation Repeat-associated non-AUG (RAN) translation drives synthesis of toxic aggregating peptides from expanded nucleotide repeat regions associated with several neurodegenerative conditions. Knockdown of DDX3X and its D. melanogaster orthologue belle were shown to suppress RAN translation selectively and strongly in transcripts containing expanded CGG repeats from the Fragile X messenger ribonucleoprotein (FMR1) transcript [bib_ref] DDX3X and specific initiation factors modulate FMR1 repeatassociated non-AUG-initiated translation, Linsalata [/bib_ref]. FMR1 RAN translation causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). In this instance, the suppressive effect of DDX3X knockdown was unrelated to start codon selection. Instead, it was suggested to be caused by stalling of ribosomes on highly structured GC-rich regions so that these were unable to reach the translation initiation site [bib_ref] DDX3X and specific initiation factors modulate FMR1 repeatassociated non-AUG-initiated translation, Linsalata [/bib_ref]. In another study,also identified DDX3X as a regulator of RAN translation. Their study investigated effects of DDX3X on translation initiation from expanded hexanucleotide GGGGCC repeats in C9ORF72 that are a cause of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia. Interestingly, the authors observed the opposite effect of DDX3X knockdown on RAN translation compared to [bib_ref] DDX3X and specific initiation factors modulate FMR1 repeatassociated non-AUG-initiated translation, Linsalata [/bib_ref]. In this case, knockdown of DDX3X enhanced RAN translation while DDX3X overexpression suppressed it, implicating DDX3X as a negative regulator of RAN-mediated translation for C9ORF72. The authors demonstrated that this was mediated via a capindependent mechanism and suggest that integrity of secondary structure elements (hairpins rather than G4 elements) formed by the GGGGCC repeats is required for RAN translation via an IRES-like mechanism. DDX3X's helicase activity was required for its suppressive effect on RAN translation, and the authors concluded that unwinding of structural elements by DDX3X impedes IRES function of the repeat region, thus blocking translation initiation. In contrast and as discussed earlier, in most instances DDX3X acts as a positive regulator of IRES-mediated translation, as has been shown for several viral and cellular IRES (Section 4.4.1). The two studies on DDX3X's role in RAN translation illustrate again that DDX3X can have complex and opposing effects on translation, likely linked to the nature and function of secondary structure elements present in specific transcripts. In this context, it is worth noting that the phenomenon of RAN translation was only described in 2011 and as such many mechanistical questions are still unanswered . It is likely that RAN translation can occur via different molecular mechanisms, including capand IRES-dependent initiation [bib_ref] Mechanisms of repeat-associated non-AUG translation in neurological microsatellite expansion disorders, Castelli [/bib_ref]. Further elucidation of the mechanistic differences between RAN translation occurring in FMR1 and C9ORF72 transcripts may help to explain the observed differential DDX3X effects. Clarifying the exact contributions of DDX3X to alternative translation initiation mechanisms should be a focus of further studies because it is likely that dysregulation of DDX3X's role in this process contributes to its involvement in disease. ## Ddx3x's role in viral translation Viral infection is a disease context in which DDX3Xmediated translation regulation plays a substantial role. DDX3X has both anti-viral functions that are targeted by viral evasion proteins, and pro-viral functions where it is co-opted by viruses to facilitate their life cycles. These roles are not mutually exclusive, i.e., DDX3X recruitment by a virus to support viral replication can simultaneously inhibit its anti-viral function. Anti-viral functions attributed to DDX3X include its capacity to form SGs as previously described (Section 4.3.1), as well as its non-conventional role as an innate immune signalling molecule in the antiviral RIG-I IFN-inducing pathway [bib_ref] Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated..., Schröder [/bib_ref] [bib_ref] Human DEAD box helicase 3 couples IκB kinase ε to interferon regulatory..., Gu [/bib_ref] [bib_ref] DDX3 directly facilitates IKKα activation and regulates downstream signalling pathways, Fullam [/bib_ref]. Pro-viral functions of DDX3X involve its ability to resolve secondary structures in viral RNAs, its ability to mediate various forms of non-canonical translation initiation (Section 4.4), as well as its ability to recruit translation machinery components (Section 3). Therefore, translation regulation likely underpins many of the proviral effects of DDX3X. As it is being considered a potential target for broad-spectrum antiviral drug development Frontiers in Cell and Developmental Biology frontiersin.org 5.1 DDX3X interacts with viral mRNAs and supports (cap-independent) translation DDX3X has been shown to associate with and support translation of a variety of viral RNAs. For example, in Japanese Encephalitis Virus infection, DDX3X is bound by viral replication complex proteins NS3 and NS5 and binds 5′ and 3′UTRs of the viral RNA to support translation and replication [bib_ref] Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral untranslated..., Li [/bib_ref]. In many viral infections, DDX3X supports viral translation in a cap-independent manner, usually via IRES-mediated translation. Cap-independent translation is a common feature of viral RNAs, as it reduces dependency on conventional translation mechanisms which can become downregulated in response to infection . In Foot and Mouth Disease Virus (FMDV) infection, DDX3X binds to an IRES in the viral RNA together with RPL13 and supports translation via recruitment of eIF3e and j [bib_ref] Ribosomal protein L13 promotes IRES-driven translation of foot-and-mouth disease virus in a..., Han [/bib_ref]. In Enterovirus 71 infection, eIF4G is truncated by viral proteases, and this processed eIF4G binds to DDX3X, presumably to recruit it for unwinding of an inhibitory stem loop within the gRNA IRES [bib_ref] Stimulation of the internal ribosome Entry site (IRES)-Dependent translation of enterovirus 71..., Su [/bib_ref]. Other IRES that display DDX3X regulation include those present in HCV [bib_ref] The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes, Geissler [/bib_ref] [bib_ref] Dynamic interaction of stress granules, DDX3X, and IKK-α mediates multiple functions in..., Pène [/bib_ref] [bib_ref] Stimulation of the internal ribosome Entry site (IRES)-Dependent translation of enterovirus 71..., Su [/bib_ref] , encephalomyocarditis virus (EMCV), Echovirus 9, and Coxsackievirus 16, clearly illustrating the extensive contribution of DDX3X-mediated non-canonical translation to viral infections [bib_ref] Stimulation of the internal ribosome Entry site (IRES)-Dependent translation of enterovirus 71..., Su [/bib_ref]. Additionally, bovine adenovirus pVIII inhibits cap-dependent translation and interacts with DDX3X. By doing so, it prevents DDX3X's association with capped mRNAs and uses it to recruit various eIFs to the viral RNA [bib_ref] Bovine adenovirus-3 pVIII suppresses cap-dependent mRNA translation possibly by interfering with the..., Ayalew [/bib_ref]. ## Viruses manipulate cytosolic granule formation by ddx3x SG formation is a common cellular response to viral infection and can arise due to interferon signalling from the anti-viral innate immune response [bib_ref] Emerging translation strategies during virus-host interaction, Hoang [/bib_ref]. As described in Section 4.3.1, SG formation has the effect of sequestering free ribosomes and RBPs that are essential for cap-dependent translation. Therefore, common viral strategies include inhibition of SG formation and usage of cap-independent IRES translation, as described above. Of note, many viruses form replication complexes and sites of translation that resemble SGs. As DDX3X is a known SG factor (Section 4.3.1), it is not surprising that it has also been shown to colocalise in SG-like cytoplasmic foci with various viral RNAs and proteins, although it is often difficult to determine whether these are genuine SGs or another type of RNA granule that promotes viral replication. By recruiting DDX3X, viruses not only acquire an RNA remodelling activity but also benefit from DDX3X's interactions with translation machinery components that it can recruit to viral RNAs (see Section 3). For example, HCV influences the cellular distribution of DDX3X over the course of infection. Early in infection, an interaction with the HCV RNA 3′UTR causes DDX3X accumulation in SGs with IKKα, Ras GTPase-activating protein-binding protein 1 (G3BP1), and PABP, which leads to IKKα activation and downstream activation of lipogenesis. 24-48 h post-infection, HCV core protein, which directly binds DDX3X [bib_ref] Hepatitis C virus core protein binds to a DEAD box RNA helicase, Mamiya [/bib_ref] [bib_ref] Hepatitis C virus core protein interacts with a human DEAD box protein..., Owsianka [/bib_ref] [bib_ref] Hepatitis C virus core protein interacts with cellular putative RNA helicase, You [/bib_ref] , recruits the helicase to lipid droplets [bib_ref] Dynamic interaction of stress granules, DDX3X, and IKK-α mediates multiple functions in..., Pène [/bib_ref]. Herpes Simplex Virus 1 (HSV-1) infection causes DDX3X to aggregate, with some degree of co-localisation with viral particles [bib_ref] The ATP-dependent RNA helicase DDX3X modulates herpes simplex virus 1 gene expression, Khadivjam [/bib_ref]. DDX3X is also recruited to viral replication complexes by murine norovirus, which displays highly structured 5′ and 3′ UTRs [bib_ref] Identification of RNA-protein interaction networks involved in the norovirus life cycle, Vashist [/bib_ref]. Many viruses actively inhibit SG formation, and some of these also interact with DDX3X. It is possible that SG induction sequesters DDX3X from sites of viral replication/translation, where it is needed by the virus. Pestivirus is an example of a virus that inhibits SG assembly and recruits DDX3X via its viral N-terminal protease [bib_ref] Host factors that interact with the pestivirus N-terminal protease, Npro, are components..., Jefferson [/bib_ref]. In West Nile Virus (WNV) infection, DDX3X localisation in cytoplasmic granules is disrupted, and it is eventually recruited to perinuclear WNV replication complexes [bib_ref] P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV..., Chahar [/bib_ref]. Finally, DDX3X has recently been identified as an important host factor in SARS-CoV-2 infection. It binds to SARS-CoV-2 RNA [bib_ref] The SARS-CoV-2 RNA-protein interactome in infected human cells, Schmidt [/bib_ref] as well as its Nucleoprotein, and is recruited to viral replication complexes along with the SG protein G3BP1 [bib_ref] Proteomic analysis identifies the RNA helicase DDX3X as a host target against..., Ciccosanti [/bib_ref]. SARS-CoV-2 infection inhibits SG formation, which releases DDX3 to promote viral translation and replication [bib_ref] Proteomic analysis identifies the RNA helicase DDX3X as a host target against..., Ciccosanti [/bib_ref]. ## Ddx3x regulates hiv-1 mrna translation HIV-1 is a well-studied example where DDX3X contributes to viral translation through cap-dependent and independent mechanisms and its ability to form RNP granules. DDX3X is an essential host factor for HIV-1 replication [bib_ref] Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function, Yedavalli [/bib_ref] and its function in translation likely underpins this requirement for DDX3X. The 5′UTR of the HIV-1 genome is a well-characterised viral RNA target of DDX3X, where it strongly binds to the stem-loop TAR motif located at the very 5′ end of the genomic RNA, although it can bind to additional downstream stem loops also [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref]. Deletion of this motif or insertion of an upstream unstructured sequence removed dependency on DDX3X for Frontiers in Cell and Developmental Biology frontiersin.org translation, suggesting that it uses its helicase activity to resolve the cap-proximal secondary structures and thereby makes the 5′ cap available for 43S PIC binding [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref]. The N-terminal domain of DDX3X may be involved in linking CRM-1-dependent nuclear export of unspliced HIV-1 gRNA with its translation and it mediates accumulation in cytoplasmic granules [bib_ref] DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the..., Frohlich [/bib_ref]. These granules contain PABP and eIF4G, but not eIF4E, suggesting the presence of a novel DDX3Xcontaining cap-binding complex which supports HIV-1 gRNA expression [bib_ref] DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs, Soto-Rifo [/bib_ref] [bib_ref] The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote..., Soto-Rifo [/bib_ref]. In addition to supporting cap-dependent translation of the HIV-1 gRNA, DDX3X also supports its cap-independent translation via an IRES located in the 5′ Long Terminal Repeat region [bib_ref] Translational regulation of HIV-1 replication by HIV-1 Rev cellular cofactors Sam68, eIF5A,..., Liu [/bib_ref]. DDX3X also enhances polysome occupancy of Tat and Rev mRNAs, suggesting that it promotes their translation. Expression of HIV-1 Tat conversely enhances expression of DDX3X, and both proteins physically interact, with DDX3X being required for recruitment of Tat to cytoplasmic granules and polysomes with viral RNA. Overall, HIV-1 appears to target DDX3X throughout infection to mediate various processes essential for the viral life cycle, employing its RNA helicase activity, interactions with eIFs, and possibly its propensity to form phase-separated RNP granules. In summary, the abilities of DDX3X to resolve RNA secondary structures, interact with the translation machinery, participate in RNA granule formation and contribute to non-canonical translation initiation make it a prime target for exploitation by a diverse range of viruses. Targeting DDX3X with inhibitors as a form of broad-spectrum anti-viral therapy holds promise, however one will need to consider its diverse range of functions and how inhibiting these affects both viral replication and host cell translation. # Conclusion An intense research effort has been made in recent years to identify DDX3X's physiological mRNA targets in different cell types, driven by a desire to understand pathophysiology of the neurodevelopmental condition DDX3X syndrome and by the prospect that DDX3X inhibitors could serve as anti-viral and anti-cancer drugs. This has significantly increased knowledge about DDX3X's role in translation regulation, yet many open questions remain: Through detailed analysis of known DDX3X binding targets, is it possible to identify determinants of DDX3X binding and translational regulation? How does DDX3X dynamically regulate translation in the context of LLPseparated RNA granules, not all of which might be conventional SGs? The mechanisms by which DDX3X regulates translation are also not fully clear, as we discussed in Section 3. Which step(s) in translation (pre-)initiation does DDX3X regulate and is this target-specific? Can DDX3X also negatively regulate translation of mRNAs aside from its role in SG formation, and if so, how? How does DDX3X interact with other RBPs to regulate translation of specific mRNA subsets? What are the consequences of DDX3X manipulation by chemical inhibitors or post-translational modifications for translation of specific mRNA target subsets? Answering this last question is particularly important as we continue to explore DDX3X's suitability as a drug target. Undoubtedly, as research interest in DDX3X continues to grow and the RNP field is seeing development of many new methodologies to analyse translation, these questions will be answered in the next few years and hopefully allow identification of strategies for manipulating DDX3X function safely in a therapeutic context. # Author contributions CR completed the systematic literature review and co-wrote the manuscript, MS supervised the process, co-wrote the manuscript and completed the final editing. [fig] FIGURE 1: Interactions between DDX3X and other RNA-binding proteins during translation (pre)-initiation. (A) Potential DDX3X-containing protein complexes associated with the mRNA 5′cap. Only known DDX3X-interacting factors are included for clarity. (i) DDX3X may be associated with CBC after nuclear export, which could allow it to contribute to pioneer translation and/or nonsense-mediated decay (NMD), or (ii) alternative translation mechanisms independent of eIF4F (Chen et al., 2018). (iii) DDX3X has also been shown to interact with eIF4G (Soto-Rifo et al., 2012) and eIF4E, meaning that it might be associated with the conventional eIF4F complex on RNA (Shih et al., 2008). (iv) Alternatively, it may form novel cap-binding complexes with PABP and eIF4G, but independently of eIF4E (Soto-Rifo et al., 2013) (B) Potential contribution of DDX3X helicase activity to recruitment of the 43S PIC to mRNA 5′ends. The presence of stem loops proximal to the 5′ cap can inhibit association of the PIC with the mRNA (left). Through destabilisation of cap-proximal structured regions on the mRNA, DDX3X may support 43S binding (right) (Soto-Rifo et al., 2013). (C) Potential contribution of DDX3X to scanning. Structured regions within the 5′UTR downstream of the scanning 48S PIC (left) have the potential to stall scanning and possibly terminate translation initiation (Abaeva et al., 2011 Gupta et al., 2018). Resolution of these structures by DDX3X may facilitate proper scanning of mRNAs containing long, structured 5′UTRs (right). [/fig] [fig] Funding: CR is a recipient of an Irish Research Council Government of Ireland Postgraduate Scholarship (GOIPG/2022/888). [/fig] [table] TABLE 1: Examples of DDX3X targets in the literature. Abbreviations: PAR-CLIP = Photoactivatable Ribonucleotide-enhanced Crosslinking and Immunoprecipitation. FISH = Fluorescence in situ hybridisation. TRAP-seq = Translating Ribosome Affinity Purification -RNAseq. Ribo-seq = Ribosome pulldown -RNAseq. LUC = Luciferase reporter translation assays. [/table]

Alcohol use in opioid agonist treatment Alcohol misuse among individuals receiving agonist treatment for an opioid use disorder is common and is associated with significant morbidity and mortality. At present, though substantial research highlights effective strategies for the screening, diagnosis and management of an alcohol or opioid use disorder individually, less is known about how best to care for those with a dual diagnosis especially since common treatments for opioid addiction may be contraindicated in a setting of alcohol use. This review summarizes existing research and characterizes the prevalence, clinical implications and management of alcohol misuse among individuals with opioid addiction. Furthermore, it highlights clinically relevant management strategies in need of future research to advance care for this unique, but important, patient population. # Background Approximately one-third of individuals who receive opioid agonist treatment (OAT), such as methadone or buprenorphine/naloxone for the management of an opioid use disorder, also misuse alcohol [bib_ref] Alcohol use disorders in opioid maintenance therapy: prevalence, clinical correlates and treatment, Soyka [/bib_ref]. Despite alcohol use being a risk factor for fatal overdose among individuals prescribed opioids, as well as being an established risk factor for addiction treatment non-compliance among OAT participants [bib_ref] Patterns of co-morbidity between alcohol use and other substance use in the..., Degenhardt [/bib_ref] [bib_ref] Buprenorphine/naloxone and methadone maintenance treatment outcomes for opioid analgesic, heroin, and combined..., Potter [/bib_ref] [bib_ref] Importance of identifying cocaine and alcohol dependent methadone clients, Rowan-Szal [/bib_ref] , little guidance currently exists outlining effective management strategies for this patient population. Consequently, an individual's alcohol misuse frequently goes undiagnosed and untreated [bib_ref] Patterns of drinking and drinking outcomes among drug misusers. 1-Year follow-up results, Gossop [/bib_ref] [bib_ref] Correlates of alcohol use among methadone patients, El-Bassel [/bib_ref] [bib_ref] Psychosocial interventions for problem alcohol use in primary care settings (PINTA): baseline..., Klimas [/bib_ref]. The potential risk for relapse to opioid use, as a result of this missed opportunity, as well as the host of negative consequences that can occur from this or from untreated alcohol misuse is significant among this patient population [bib_ref] Anger and depressive states among treatment-seeking drug abusers: testing the psychopharmacological specificity..., Aharonovich [/bib_ref] [bib_ref] Comparison of opiate-primary treatment seekers with and without alcohol use disorder, Hartzler [/bib_ref] [bib_ref] The relationship between lifetime abuse and suicidal ideation in a sample of..., Lloyd [/bib_ref] [bib_ref] Role of alcohol in the progression of liver disease caused by hepatitis..., Ostapowicz [/bib_ref] [bib_ref] Self-injurious behaviour, traumatic life events and alexithymia among treatment-seeking opiate addicts: prevalence,..., Oyefeso [/bib_ref] [bib_ref] Assessment of long-term outcomes of community-acquired hepatitis C infection in a cohort..., Rodger [/bib_ref] [bib_ref] Excessive alcohol consumption is associated with reduced quality of life among methadone..., Senbanjo [/bib_ref] [bib_ref] Intravenous opiate maintenance in a cohort of injecting drug addicts, Sendi [/bib_ref]. This review summarizes the existing research of alcohol misuse among OAT participants with a specific focus on prevalence, clinical implications and management. Clinically relevant management strategies in need of future research are additionally highlighted to advance care for this unique, but important, patient population. # Methods ## Search strategy This narrative review was based on a literature search using Pubmed and Ovid Medline databases. Keywords used described unhealthy patterns of alcohol use and included: alcohol, alcohol addiction, alcohol misuse, harmful alcohol use, hazardous alcohol use, heavy alcohol use, alcohol abuse, alcohol dependence or alcohol use disorder. These terms were combined with terms referring to OAT including: opioid addiction treatment, OAT, buprenorphine or methadone. Studies written in English were included. Additionally, references for all studies identified through the database search were examined to identify articles that may have been missed. Articles focused on prevalence, clinical implications, screening or management of alcohol misuse among OAT participants were reviewed in detail and are summarized. ## Prevalence Estimating the prevalence of alcohol misuse among opioid dependent individuals receiving OAT is challenging. Substantial variation exists within the literature among patient populations and treatment settings being studied. Furthermore a lack of standardization pertaining to alcohol misuse terminology and measurement of this is common. In this review, 'alcohol misuse' is defined as the consumption of alcohol in a quantity that exceeds low risk for developing an alcohol use disorder as defined by the National Institute on Alcohol Abuse and Alcoholism (i.e. no more than 3 drinks in a single day and no more than 7 drinks per week for women and no more than 4 drinks in a single day and no more than 14 drinks per week for men) and includes both people with 'risky drinking, ' 'alcohol abuse or dependence' and those with an established 'alcohol use disorder'. A 2015 review by Soyka et al., estimated one-third of methadone maintenance participants also have problematic alcohol use [bib_ref] Alcohol use disorders in opioid maintenance therapy: prevalence, clinical correlates and treatment, Soyka [/bib_ref]. Other studies are in agreement with this estimate including a meta-analyses of U.S. clinical trials which demonstrated 38% of individuals seeking treatment for opioid use to have a concurrent alcohol use disorder, as defined by a diagnosis of either alcohol abuse or dependence using criteria from the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) [bib_ref] Comparison of opiate-primary treatment seekers with and without alcohol use disorder, Hartzler [/bib_ref]. Beyond the U.S., data from the British National Treatment Outcome Research Study, a large prospective study of drug users, indicates that at the time of enrolment in a community methadone clinic just over one-third of clients were drinking alcohol above the recommended limits with no statistically significant change observed after 1 year of follow-up [bib_ref] Patterns of drinking and drinking outcomes among drug misusers. 1-Year follow-up results, Gossop [/bib_ref]. Lastly, a cross-sectional study of current or former heroin users attending primary care for methadone maintenance treatment in Ireland revealed the prevalence of problem alcohol use [as defined by an Alcohol Use Disorder Identification Test (AUDIT) score of >7] to be 35% [bib_ref] Prevalence of problem alcohol use among patients attending primary care for methadone..., Ryder [/bib_ref]. Collectively, these data suggest that approximately one-third of opioid addicted individuals in treatment may have concurrent alcohol misuse. ## Interactions While interactions between alcohol and opioids have previously been described [bib_ref] Alcohol use disorders in opioid maintenance therapy: prevalence, clinical correlates and treatment, Soyka [/bib_ref] [bib_ref] Opioids and alcoholism, Oswald [/bib_ref] , research focused specifically on alcohol in the context of OAT is scarce [bib_ref] Effect of opioid substitution therapy on alcohol metabolism, Clark [/bib_ref]. Animal studies involving methadone predominate and repeatedly demonstrate an influence of ethanol on methadone metabolism and vice versa [bib_ref] Effect of opioid substitution therapy on alcohol metabolism, Clark [/bib_ref]. More specifically, among rat subjects, acute ethanol consumption increased peak methadone concentration [bib_ref] Evaluation of the methadone-alcohol interaction. I. Alterations of plasma concentration kinetics, Donnelly [/bib_ref] [bib_ref] Alcohol and the steady-state disposition kinetics of methadone in rats, Tommasello [/bib_ref] while chronic ethanol use led to a reduction in peak methadone levels [bib_ref] Alcohol and the steady-state disposition kinetics of methadone in rats, Tommasello [/bib_ref] [bib_ref] Interaction of methadone and ethanol metabolism, Borowsky [/bib_ref] [bib_ref] Metabolic interactions between opiates and alcohol, Kreek [/bib_ref]. Similarly, acute methadone administration has been shown to decrease the rate of ethanol metabolism (and thus increase blood alcohol levels) [bib_ref] Evaluation of the methadone-alcohol interaction. I. Alterations of plasma concentration kinetics, Donnelly [/bib_ref] whereas chronic methadone use leads to a reduction in blood alcohol levels [bib_ref] Effect of ethanol concentration on rates of ethanol elimination in normal rats..., Wendell [/bib_ref] [bib_ref] Effects of chronic methadone treatment on the rate of ethanol elimination in..., Umans [/bib_ref]. Human research focused on this issue is scarce [bib_ref] Effect of opioid substitution therapy on alcohol metabolism, Clark [/bib_ref]. Clinical observations among individuals who receive methadone maintenance therapy report less of an effect of alcohol [bib_ref] Metabolic interactions between opiates and alcohol, Kreek [/bib_ref] [bib_ref] Opioid interactions with alcohol, Kreek [/bib_ref] , more sedation at the time of peak methadone levels as well as more rapid dissipation of methadone's overall effect resulting in opioid withdrawal symptoms [bib_ref] Metabolic interactions between opiates and alcohol, Kreek [/bib_ref]. One study by Lenne et al. [bib_ref] The effects of the opioid pharmacotherapies methadone, LAAM and buprenorphine, alone and..., Lenne [/bib_ref] did demonstrate a small but significant effect of increased blood alcohol concentration (BAC) among non-opioid study controls compared to those receiving regular OAT. A subsequent study by Clark et al. [bib_ref] Effect of opioid substitution therapy on alcohol metabolism, Clark [/bib_ref] furthered these findings by demonstrating the interaction between alcohol and opioids to be strongest at the time of peak plasma levels after opioid dosing (i.e. a dose-response relationship) as well as reporting an opioid-specific difference in the magnitude of this interaction (e.g. methadone versus buprenorphine). While these findings support the case for a true pharmacokinetic interaction among humans between alcohol and opioids, the specific site(s) of such interaction requires further study. Furthermore, it should be emphasized that the overall magnitude of the reduction of BAC among individuals receiving OAT in these studies is small (and likely of limited clinical significance) and individuals who receive OAT and consume alcohol will still experience a greater opioid effect due to the combined sedative effect of both substances [bib_ref] Effect of opioid substitution therapy on alcohol metabolism, Clark [/bib_ref]. ## Clinical implications Knowledge of the potential mechanisms of interaction between OAT and alcohol and their effect on blood levels is of importance, but equally so is determining the clinical significance of these mechanisms. ## Effect of oat initiation on alcohol consumption To date, studies investigating the effect of OAT initiation on alcohol consumption among individuals with alcohol misuse and an opioid use disorder are mixed. For example, Caputo et al. [bib_ref] Short-term methadone administration reduces alcohol consumption in non-alcoholic heroin addicts, Caputo [/bib_ref] demonstrated short term methadone treatment to be associated with a reduction in alcohol levels while long term methadone maintenance therapy resulted in increased alcohol consumption. While an inverse relationship between heroin use and alcohol use has previously been described [bib_ref] Longitudinal patterns of alcohol use by narcotics addicts, Hser [/bib_ref] [bib_ref] Alcohol use by heroin addicts: evidence for an inverse relationship. A study..., Anglin [/bib_ref] , a recent study found methadone enrolment to have no effect on heavy drinking and may even appear to decrease the initiation of heavy drinking among heroin users [bib_ref] The impact of enrolment in methadone maintenance therapy on initiation of heavy..., Klimas [/bib_ref]. Furthermore, a 12-month longitudinal study of individuals with both heroin addiction and alcohol dependence demonstrated both methadone and buprenorphine to be associated with a reduction in alcohol use, with buprenorphine being more efficacious [bib_ref] Opioid maintenance therapy suppresses alcohol intake in heroin addicts with alcohol dependence:..., Nava [/bib_ref]. Lastly, a recent meta-analyses involving 15 studies showed no clear pattern regarding the effects of OAT on alcohol consumption with 3 studies indicating an increase in alcohol consumption during treatment, 3 studies indicating a decrease in alcohol consumption and 9 studies reporting no change [bib_ref] The effect of methadone maintenance treatment on alcohol consumption: a systematic review, Srivastava [/bib_ref]. ## Overdose and mortality Alcohol use has previously been identified to be a risk factor for increased overdose and mortality among individuals receiving OAT [bib_ref] Deaths in methadone maintenance treatment in New South Wales, Zador [/bib_ref] [bib_ref] Alcoholism and methadone treatment: consequences for the patient and program, Joseph [/bib_ref]. The degree of increased risk conferred overall and according to alcohol consumption patterns however (i.e. hazardous or harmful use compared to an alcohol use disorder) is currently lacking. A cross-sectional study by Zador et al. examined the number and causes of death among participants of a methadone maintenance treatment program in Australia and demonstrated drug-related death to account for the highest proportion of mortality (44%), with alcohol use being cited as the third most common substance of use after benzodiazepines and other opioids [bib_ref] Deaths in methadone maintenance treatment in New South Wales, Zador [/bib_ref]. Similarly, a New-York based longitudinal follow-up study of active and discharged methadone patients reported excessive alcohol use (≥4 oz per day for a 3 month period) to be the leading cause of death among active methadone participants (35%) and the second most common cause of death, following complications with opiates, among discharged methadone patients (39%) [bib_ref] Alcoholism and methadone treatment: consequences for the patient and program, Joseph [/bib_ref]. Mechanisms driving this process are likely diverse (e.g. suicide attempts, unintentional overdoses involving various substances including benzodiazepines, illicit opioids and OAT, etc.) and not well described, but likely relate to the interactions between alcohol and methadone outlined previously. As such, individuals should routinely be advised of the compounded risk of acute and chronic alcohol consumption while in receipt of OAT and, in particular, of the risk of relapse to illicit opioid use. In addition, during methadone initiation, a period already known to be associated with an increased risk for overdose and mortality [bib_ref] Deaths in methadone maintenance treatment in New South Wales, Zador [/bib_ref] [bib_ref] Mortality associated with New South Wales methadone programs in 1994: lives lost..., Caplehorn [/bib_ref] , concurrent acute ethanol consumption can further compound this risk by increasing CNS and/or respiratory depression [bib_ref] An analysis of the root causes for opioid-related overdose deaths in the..., Webster [/bib_ref] [bib_ref] Comparison of fatal poisonings by prescription opioids, Hakkinen [/bib_ref]. Similarly, though methadone maintenance treatment may lead to lower blood alcohol levels after consumption compared to non-methadone users, one's overall risk for overdose and mortality is still increased given the combined sedative effect of both methadone and alcohol [bib_ref] Opioid interactions with alcohol, Kreek [/bib_ref]. ## Other clinical outcomes Beyond increasing one's risk for overdose and mortality, alcohol misuse among individuals concurrently receiving OAT has been associated with a host of other negative clinical outcomes. Specific to addiction treatment, alcohol misuse has been shown to be risk factor for poor compliance with pharmacotherapy [bib_ref] Comparison of opiate-primary treatment seekers with and without alcohol use disorder, Hartzler [/bib_ref] and a predictor for negative treatment outcomes [bib_ref] Patterns of co-morbidity between alcohol use and other substance use in the..., Degenhardt [/bib_ref] [bib_ref] Buprenorphine/naloxone and methadone maintenance treatment outcomes for opioid analgesic, heroin, and combined..., Potter [/bib_ref] [bib_ref] Importance of identifying cocaine and alcohol dependent methadone clients, Rowan-Szal [/bib_ref]. As such, individuals with ongoing alcohol misuse are at an increased risk for a relapse to opioids or other substances [bib_ref] Comparison of opiate-primary treatment seekers with and without alcohol use disorder, Hartzler [/bib_ref]. Furthermore, as hepatitis infection is a common comorbidity among opioid dependent individuals with prevalence estimates ranging from 64 to 100% in some cohorts [bib_ref] Seroprevalence of hepatitis C in a sample of middle class substance abusers, Abraham [/bib_ref] [bib_ref] Factors associated with prevalent hepatitis C: differences among young adult injection drug..., Diaz [/bib_ref] [bib_ref] Age-specific seroprevalence of HIV, hepatitis B virus, and hepatitis C virus infection..., Murrill [/bib_ref] [bib_ref] Critical issues in the treatment of hepatitis C virus infection in methadone..., Novick [/bib_ref] [bib_ref] Incidence of hepatitis C virus infection among injection drug users during an..., Patrick [/bib_ref] [bib_ref] Hepatitis C and substance use in a sample of homeless people in..., Rosenblum [/bib_ref] , chronic alcohol misuse can result in hepatotoxicity and increase an individual's risk for progression to cirrhosis [bib_ref] Role of alcohol in the progression of liver disease caused by hepatitis..., Ostapowicz [/bib_ref] [bib_ref] Assessment of long-term outcomes of community-acquired hepatitis C infection in a cohort..., Rodger [/bib_ref] [bib_ref] Intravenous opiate maintenance in a cohort of injecting drug addicts, Sendi [/bib_ref]. Additionally, alcohol misuse can exacerbate psychiatric comorbidities such as anxiety, depression and suicidality, all of which are known to be more common among OAT recipients [bib_ref] Anger and depressive states among treatment-seeking drug abusers: testing the psychopharmacological specificity..., Aharonovich [/bib_ref] [bib_ref] The relationship between lifetime abuse and suicidal ideation in a sample of..., Lloyd [/bib_ref] [bib_ref] Self-injurious behaviour, traumatic life events and alexithymia among treatment-seeking opiate addicts: prevalence,..., Oyefeso [/bib_ref] [bib_ref] Comorbid psychopathology and alcohol use patterns among methadone maintenance treatment patients, Moussas [/bib_ref]. Lastly, a study by Sebanjo et al. demonstrated alcohol misuse to be associated with a significant reduction in quality of life and social functioning among methadone maintained individuals [bib_ref] Excessive alcohol consumption is associated with reduced quality of life among methadone..., Senbanjo [/bib_ref]. ## Screening Annual screening and brief intervention for alcohol misuse among OAT participants is recommended by clinical guidelines given both its prevalence and potential for a myriad of negative consequences. While the effectiveness of such practices among general populations has shown mixed results [bib_ref] Brief intervention for problem drug use in safety-net primary care settings: a..., Roy-Byrne [/bib_ref] [bib_ref] Screening and brief intervention for drug use in primary care: the ASPIRE..., Saitz [/bib_ref] [bib_ref] Brief interventions for heavy alcohol users admitted to general hospital wards, Mcqueen [/bib_ref] , a significant reduction in alcohol consumption has been observed among methadone maintenance participants in several trials including part of a systematic review [bib_ref] Brief interventions are effective in reducing alcohol consumption in opiatedependent methadone-maintained patients:..., Darker [/bib_ref] [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] [bib_ref] Psychosocial interventions to reduce alcohol consumption in concurrent problem alcohol and illicit..., Klimas [/bib_ref] [bib_ref] Helping methadone patients who drink excessively to drink less: short-term outcomes of..., Bennett [/bib_ref] [bib_ref] Screening and brief interventions for illicit drug use and alcohol use in..., Darker [/bib_ref]. More specifically, these studies included participants of both community and designated methadone maintenance clinics in both a European [bib_ref] Brief interventions are effective in reducing alcohol consumption in opiatedependent methadone-maintained patients:..., Darker [/bib_ref] [bib_ref] Helping methadone patients who drink excessively to drink less: short-term outcomes of..., Bennett [/bib_ref] and U.S. [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] setting with the intervention being delivered by either a clinician [bib_ref] Brief interventions are effective in reducing alcohol consumption in opiatedependent methadone-maintained patients:..., Darker [/bib_ref] , nurse [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] [bib_ref] Helping methadone patients who drink excessively to drink less: short-term outcomes of..., Bennett [/bib_ref] or trained therapist [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref]. Despite this finding, implementation of these interventions among primary care providers of OAT has been slow [bib_ref] Problem alcohol use among problem drug users in primary care: a qualitative..., Field [/bib_ref] and remains variable with rates ranging between 2 and 93% [bib_ref] Strategies to implement alcohol screening and brief intervention in primary care settings:..., Williams [/bib_ref] [bib_ref] Engaging general practitioners in the management of hazardous and harmful alcohol consumption:..., Anderson [/bib_ref]. Furthermore, when screening does occur in these settings it is often completed without the use of a validated screening tool [bib_ref] Psychosocial interventions for problem alcohol use in primary care settings (PINTA): baseline..., Klimas [/bib_ref]. Suggested reasons for these findings identify time restrictions, lack of resources and physician attitudes about the effectiveness of screening and brief intervention for the detection and management of alcohol misuse [bib_ref] Psychosocial interventions to reduce alcohol consumption in concurrent problem alcohol and illicit..., Klimas [/bib_ref]. Creation of a guideline for alcohol misuse screening and treatment specific for OAT participants has previously been described as a potential solution to mitigate these challenges [bib_ref] Problem alcohol use among problem drug users: development and content of clinical..., Klimas [/bib_ref]. Reasons for such a document include: (1) the high prevalence of alcohol misuse among OAT participants, suggesting the need for a more proactive and systematic approach to screening and treatment; (2) consideration for the use of lower thresholds to not only define alcohol misuse but also guide timing for referral to treatment; and (3) the need for involvement of an addiction specialist for severe cases of recurrent or persistent alcohol misuse among this patient population. While no such dedicated guideline exists in the U.S., a recent clinical guideline was published in Europe and addressed problem alcohol use among substance users who attended primary care (the vast majority for OAT) in Ireland [bib_ref] Problem alcohol use among problem drug users: development and content of clinical..., Klimas [/bib_ref]. Screening recommendations from this guideline suggest random, but at least annual, screening for alcohol misuse using AUDIT C (a 3-item version of the Alcohol Use Disorder Identification Test) as an initial screening instrument, with a positive result requiring administration of the full AUDIT. While other diagnostic screening tools (e.g. blood or urine tests, breathalyzer) can be incorporated into the screening process, their utility is limited and should be reserved to either provide pertinent information to a treating physician or help motivate a patient to address their alcohol misuse. A positive screening test for alcohol misuse should be followed up with screening for other substance use and medical comorbidities including hepatitis and other chronic diseases (e.g. cardiac, liver). ## Management Despite one-third of OAT participants misusing alcohol, treatment for this has widely been ignored [bib_ref] Alcohol use disorders in opioid maintenance therapy: prevalence, clinical correlates and treatment, Soyka [/bib_ref]. One New York study demonstrated 21% of methadone maintenance patients to misuse alcohol with only 5% being enrolled in outpatient alcohol detox and 7% engaging in psychosocial intervention [bib_ref] Correlates of alcohol use among methadone patients, El-Bassel [/bib_ref]. A more recent 12-month follow up study among people who use drugs demonstrated little improvement in patterns of drinking among the majority of participants [bib_ref] Patterns of drinking and drinking outcomes among drug misusers. 1-Year follow-up results, Gossop [/bib_ref]. These findings may be explained by the limited access to alcohol treatment programs that exists for this patient population given many such programs require termination of a patient's OAT use as a condition of acceptance [bib_ref] Correlates of alcohol use among methadone patients, El-Bassel [/bib_ref]. To date, though a theoretical risk for over sedation or overdose may exist among OAT participants being treated for alcohol withdrawal, no research to date has clearly quantified the magnitude of this risk or demonstrated any clear interaction between sedative medications used during alcohol detoxification or treatment (e.g. benzodiazepines, barbiturates) and OAT regarding these specific outcomes (though a precipitated opioid withdrawal syndrome has previously been reported with concurrent methadone and phenobarbital administration) [bib_ref] Methadone: a review of drug-drug and pathophysiological interactions, Kapur [/bib_ref]. This may relate to the use of these medications within a therapeutic dose range and their administration, which often occurs in a supervised setting. As such, at the present time, no justifiable clinical reason exists to deny entry for treatment of alcohol misuse to an individual that is well established on an OAT program or the need for any modification in dose. Doing so may only increase one's risk for opioid relapse and the host of negative medical and psychosocial consequences as described above. Based on the above, all OAT participants identified as having alcohol misuse should be offered treatment. In the acute period, management of alcohol withdrawal in an effective and safe manner is the most important consideration. Unfortunately strategies on how best to accomplish this are lacking in the literature and warrant further study. Validation of risk scoring tools like the Prediction of Alcohol Withdrawal Severity Scale (PAWSS) among this patient population may be of benefit to identify individuals who are at low risk of developing severe, complicated alcohol withdrawal and thus do not require inpatient admission or benzodiazepine therapy for symptom management [bib_ref] Prospective validation study of the prediction of alcohol withdrawal severity scale (PAWSS)..., Maldonado [/bib_ref]. Psychosocial interventions for alcohol misuse among OAT participants have previously been described [bib_ref] Brief interventions are effective in reducing alcohol consumption in opiatedependent methadone-maintained patients:..., Darker [/bib_ref] [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] [bib_ref] Helping methadone patients who drink excessively to drink less: short-term outcomes of..., Bennett [/bib_ref]. More specifically, clinician delivered brief intervention was shown to reduce alcohol consumption among OAT participants without alcohol use disorders (AUDIT score <20). Such a treatment approach is recommended for all alcohol misusers identified through screening by the European clinical guidelines previously described [bib_ref] Problem alcohol use among problem drug users: development and content of clinical..., Klimas [/bib_ref]. Furthermore a pilot study and randomized controlled trial have identified motivational interviewing to be an effective strategy to reduce alcohol consumption among alcohol misusing methadone maintained participants [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] [bib_ref] Helping methadone patients who drink excessively to drink less: short-term outcomes of..., Bennett [/bib_ref]. Though not specific to OAT participants, psychosocial interventions for alcohol misuse among concurrent substance users have been described in a systematic review [bib_ref] Psychosocial interventions to reduce alcohol consumption in concurrent problem alcohol and illicit..., Klimas [/bib_ref]. Four studies involving 594 participants evaluated 6 psychosocial interventions through 4 comparison groups: cognitive-behavioral coping skills training versus 12-step facilitation (n = 41) [bib_ref] Treatment of cocaine and alcohol dependence with psychotherapy and disulfiram, Carroll [/bib_ref] , brief intervention versus treatment as usual (n = 110) [bib_ref] Alcohol-related brief intervention in patients treated for opiate or cocaine dependence: a..., Feldman [/bib_ref] , hepatitis health promotion versus motivational interviewing (n = 256) [bib_ref] Effect of motivational interviewing on reduction of alcohol use, Nyamathi [/bib_ref] and brief motivational intervention versus assessment only group (n = 187) [bib_ref] A randomized trial of a brief alcohol intervention for needle exchangers (BRAINE), Stein [/bib_ref]. Higher rates of decreased alcohol use were found at 3 and 9 months among the treatment as usual group when compared to brief intervention [bib_ref] Alcohol-related brief intervention in patients treated for opiate or cocaine dependence: a..., Feldman [/bib_ref] and more people reduced their alcohol use at 6 months (by 7 or more days in the preceding 30 days) in the brief motivational intervention group compared to control [bib_ref] A randomized trial of a brief alcohol intervention for needle exchangers (BRAINE), Stein [/bib_ref]. No other comparisons were found to be statistically significant and because of methodological study differences, no meta-analysis could be performed. Overall the authors were unable to recommend for or against the use of psychosocial interventions for alcohol misuse among concurrent substance users, which is similar to previous findings [bib_ref] Treatment of co-occurring alcohol and other drug use disorders, Arias [/bib_ref] [bib_ref] The treatment of alcoholic methadone patients: a review, Bickel [/bib_ref]. While the effectiveness of medications for alcohol relapse prevention including naltrexone and acamprosate has been described among the general population [bib_ref] Opioid antagonists for alcohol dependence, Rosner [/bib_ref] [bib_ref] Acamprosate for alcohol dependence, Rosner [/bib_ref] , opioid antagonist use among those on OAT is not possible given the effects of naltrexone on OAT and the emergence of precipitated withdrawal. In terms of acamprosate, no studies to date have been conducted among OAT participants who misuse alcohol. The use of disulfiram for reducing heavy alcohol consumption among patients receiving methadone maintenance therapy was evaluated in one randomized double-blind controlled trial [bib_ref] Use of disulfiram for alcoholics in methadone maintenance programs. A Veterans Administration..., Ling [/bib_ref] with the results showing no significant difference compared to placebo (though the trial was stopped early when sample size targets were not achieved). While two subsequent meta-analyses [bib_ref] The efficacy of disulfiram for the treatment of alcohol use disorder, Jorgensen [/bib_ref] [bib_ref] Disulfiram efficacy in the treatment of alcohol dependence: a meta-analysis, Skinner [/bib_ref] did demonstrate efficacy with the use of disulfiram when administered in a supervised setting among individuals with alcohol abuse or dependence regarding short term abstinence, days until relapse and number of drinking days, it should be noted that receipt of methadone was an exclusion criteria in one of these studies [bib_ref] The efficacy of disulfiram for the treatment of alcohol use disorder, Jorgensen [/bib_ref] with the other [bib_ref] Disulfiram efficacy in the treatment of alcohol dependence: a meta-analysis, Skinner [/bib_ref] including only 2 small randomized controlled trials of methadone maintenance patients. Given these findings, there is an urgent need to evaluate the use of such medications, or others used off label for the treatment of alcohol addiction (e.g. gabapentin) [bib_ref] A double-blind trial of gabapentin versus lorazepam in the treatment of alcohol..., Myrick [/bib_ref] [bib_ref] Gabapentin versus chlordiazepoxide for outpatient alcohol detoxification treatment, Stock [/bib_ref] specifically among OAT participants, as their administration in this setting is a feasible strategy. Another promising option for this treatment population is extended release naltrexone. Here, while oral naltrexone has not been shown to be superior to placebo in the context of opioid dependence, studies of extended release naltrexone (XR-NTX) have shown promise for the treatment of both alcohol misuse and opioid dependence [bib_ref] Efficacy and tolerability of long-acting injectable naltrexone for alcohol dependence: a randomized..., Garbutt [/bib_ref] [bib_ref] Injectable, sustained-release naltrexone for the treatment of opioid dependence: a randomized, placebo-controlled..., Comer [/bib_ref] [bib_ref] Injectable extended-release naltrexone for opioid dependence: a double-blind, placebo-controlled, multicentre randomised trial, Krupitsky [/bib_ref]. Prior to initiation with this opioid antagonist, patients are required to have completed opioid detoxification and not be receiving any ongoing opioids (including either methadone or buprenorphine). In settings where XR-NTX is available, this would be an option and its rigorous evaluation in the context of alcohol and opioid poly-substance addiction is warranted. # Conclusions Alcohol misuse is common among OAT participants and is associated with a number of adverse outcomes including overdose and mortality. Despite this, the literature suggests that screening and treatment for alcohol misuse among this patient population consistently goes overlooked. To overcome these challenges, future research should focus on the development of strategies to increase rates and frequency of alcohol screening and brief intervention among OAT providers. Guidance for effective alcohol detoxification strategies and an evaluation of acamprosate's and XR-NTX's effectiveness for relapse prevention among this patient population is also of importance. Lastly, eliminating barriers for accessing alcohol addiction treatment programs for individuals on OAT is essential as is the integration of alcohol misuse treatment into OAT primary care settings. Authors' contributions SN and EW designed and prepared the first draft of the manuscript. All coauthors contributed to drafting the final manuscript. All authors read and approved the final manuscript. ## Author details [table] 1: British Columbia Centre for Excellence in HIV/AIDS, St. Paul's Hospital, 608-1081 Burrard Street, Vancouver, BC V6Z 1Y6, Canada. 2 Department of Medicine, University of British Columbia, St. Paul's Hospital, 608-1081 Burrard Street, Vancouver, BC V6Z 1Y6, Canada. 3 School of Medicine and Medical Science, University College Dublin, Coombe Healthcare Centre, Dolphins Barn, Dublin, Ireland. [/table]

Rhein for treating diabetes mellitus: A pharmacological and mechanistic overview With the extension of life expectancy and changes in lifestyle, the prevalence of diabetes mellitus is increasing worldwide. Rheum palmatum L. a natural botanical medicine, has been used for thousands of years to prevent and treat diabetes mellitus in Eastern countries. Rhein, the main active component of rhubarb, is a 1, 8dihydroxy anthraquinone derivative. Previous studies have extensively explored the clinical application of rhein. However, a comprehensive review of the antidiabetic effects of rhein has not been conducted. This review summarizes studies published over the past decade on the antidiabetic effects of rhein, covering the biological characteristics of Rheum palmatum L. and the pharmacological effects and pharmacokinetic characteristics of rhein. The review demonstrates that rhein can prevent and treat diabetes mellitus by ameliorating insulin resistance, possess anti-inflammatory and anti-oxidative stress properties, and protect islet cells, thus providing a theoretical basis for the application of rhein as an antidiabetic agent. (2023), Rhein for treating diabetes mellitus: A pharmacological and mechanistic overview.Rheum palmatum L. is a perennial herb that belongs to the Polygonaceae family. There are approximately 60 species worldwide, mainly distributed on mountain slopes or valley wetlands at an altitude of 1,500-4,400 m. The center of distribution of this genus is China, with 41 species and four varieties, accounting for approximately 75% of the total plants in the genus. Rhubarb has been used in medicine for over 1,000 years. The traditional Chinese medicine (TCM) theory holds that rhubarb is bitter and cold and has the function of reducing accumulation, clearing heat, and eliminating fire; cooling and detoxifying blood; and removing blood stasis and meridian obstruction(Peigen et al., 1984). The detailed characteristics of Rheum palmatum L. and rhubarb are shown inFigure 1.Botanical characteristicsRheum palmatum L. is a tall stout herb, 1.5-2.0 m high, with thick woody roots and rhizomes. The stem is upright and hollow, and the leaves are nearly equal in length and width, up to 40-60 cm long. The apex is narrow and pointed, and the base is nearly heart-shaped, usually palmately hemi-5-lobed. The petiole is stout, cylindrical, nearly equal in length to the leaf, and densely covered with rusty papillary hairs. Cauline leaves get smaller upwards, while the stalks get shorter. The leaf sheath is large, up to 15 cm long, with a smooth inner surface and a rough exterior. The inflorescences are large, conical, branched, and more clustered, densely covered with coarse short hairs. The flowers are small, usually purple-red and sometimes yellowish white. The peduncle is 2.0-2.5 mm long, and the joints are located below the middle. The fruit is rounded, oval to rectangular, 8.0-9.0 mm long, and 7.0-7.5 mm wide. The seeds are broadly ovate and brownish black. The flowering period is June, and the fruiting period is August. Rhubarb is mainly distributed in the temperate and subtropical regions of Asia. # Introduction The diabetes mellitus (DM) epidemic is a global public health problem that imposes a serious social and economic burden. According to the International Diabetes Federation, DM affects an estimated 537 million adults (aged 20-79 years) worldwide, resulting in 6.7 million deaths . The national cost of DM in the United States (US) in 2017 was more than $327 billion, up from $245 billion in 2012 (American Diabetes Association, 2018). Hyperglycemia can cause multiple complications, such as diabetic retinopathy, diabetic nephropathy, atherosclerosis, cardiovascular disease, obesity, hypertension, hyperlipidemia, cerebrovascular disease, skin complications, depression, and ultimately death. The United States National Vital Statistics System stated that DM was the 8TH leading cause of death in the United States in 2020 [bib_ref] Provisional mortality data -United States, Ahmad [/bib_ref]. Timely control of blood sugar with effective medications is important. In addition to traditional antidiabetic drugs and insulin therapy, natural compounds isolated from herbal medicines have unique advantages and great potential in preventing and treating DM [bib_ref] Research progress on the mechanism of single-Chinese medicinal herbs in treating diabetes..., Yang [/bib_ref]. The World Ethnobotanical Inspection reports approximately 800 plant species that are used to treat DM [bib_ref] Ethnobotanical study of medicinal plants from Ghana;confirmation of ethnobotanical uses, and review..., Deniyi [/bib_ref]. Rhubarb is the root and rhizome of Rheum palmatum L. It has been widely used for treating obesity [bib_ref] Effect of isgin (Rheum ribes L.) on biochemical parameters, antioxidant activity and..., Bati [/bib_ref] , DM [bib_ref] Ameliorative effect and mechanism of the purified anthraquinone-glycoside preparation from rheum palmatum..., Cheng [/bib_ref] , chronic kidney disease [bib_ref] Chrysophanol ameliorates renal interstitial fibrosis by inhibiting the TGF-β/Smad signaling pathway, Dou [/bib_ref] , pneumonia [bib_ref] Rhein suppresses lung inflammatory injury induced by human respiratory syncytial virus through..., Shen [/bib_ref] , osteoarthritis [bib_ref] Emodin ameliorates cartilage degradation in osteoarthritis by inhibiting NF-κB and Wnt/β-catenin signaling..., Ding [/bib_ref] , and constipation , among others. Rhein (4,5-dihydroxyanthraquinone-2carboxylic acid) is an anthraquinone compound extracted from rhubarb, a traditional Chinese medicine. Rhein is commonly used to treat DM in Asian and European countries. The aim of this review was to elucidate the antidiabetic effects and pharmacological mechanisms of rhein. Biological characteristics of Rheum palmatum L The main therapeutic ingredient of rhubarb Rhubarb contains a variety of biologically active ingredients, such as anthraquinone derivatives, anthracene derivatives, styrene, tannins, acyl glycosides, chromones, and phenylbutazone glycosides (Pharmacopoeia of the People's Republic of . However, the main ones are rhein, emodin, aloe-emodin, chrysophanol, and emodin methyl ether [bib_ref] Analysis of phenolic compounds in rhubarbs using liquid chromatography coupled with electrospray..., Ye [/bib_ref]. The chemical structures of these compounds are depicted in . The extraction methods for the active ingredients include marinated extraction, heat reflux extraction (HRE), Soxhlet extraction (SE), and microwave-assisted extraction (MAE) [bib_ref] Application of non-ionic surfactant in the microwaveassisted extraction of alkaloids from Rhizoma..., Sun [/bib_ref] [bib_ref] Optimised ultrasonic-assisted extraction of flavonoids from Folium eucommiae and evaluation of antioxidant..., Huang [/bib_ref]. In the 21st century, rhubarb has been used in various medicinal and edible applications. There is increasing evidence that rhein is effective against DM and its complications and can act on different antidiabetic drug targets, indicating its potential as an anti-diabetic agent [bib_ref] Anti-diabetic agents from natural products--an update from, Qi [/bib_ref] [bib_ref] The molecular mechanism of rhein in diabetic nephropathy, Zeng [/bib_ref] [bib_ref] Naturally occurring anthraquinones: Chemistry and therapeutic potential in autoimmune diabetes, Chien [/bib_ref]. ## Extraction methods and quality control The extraction methods of rhein include ultrasound-assisted extraction (UAE), HRE SE, and MAE [bib_ref] Application of non-ionic surfactant in the microwaveassisted extraction of alkaloids from Rhizoma..., Sun [/bib_ref] [bib_ref] Optimised ultrasonic-assisted extraction of flavonoids from Folium eucommiae and evaluation of antioxidant..., Huang [/bib_ref]. The UAE method has the advantages of high extraction Plant and Slice of root of Rheum palmatum L. The leaves of Rheum palmatum L. are palmately lobed to half-lobed, with the lobes mostly narrowly triangular; its flowers are small, purple-red or yellowish-white; its buds are inverted pyramidal; and its fruits are small. The outer skin of rhubarb is yellowishbrown or reddish-brown, with white-like reticulate texture and scattered stars, and its broken surface is light reddish-brown or yellowish-brown, showing granularity; it has a fresh aroma and a bitter and slightly astringent taste. (A) Rheum palmatum L. is a kind of herb with medicinal value. (B) prepared slices of Rhubarb, containing a variety of active pharmacological ingredients. Frontiers in Pharmacology frontiersin.org 02 efficiency, short consumption time, and low temperature. The HRE method is suitable for small amounts of drugs that aren't easily destroyed by heat. The SE method is easy to operate, and the MAE method has the characteristics of uniform and selective heating. In recent years, proton ionic liquids have been used as efficient solvents for MAE of rhein . A variety of qualitative and quantitative analytical methods have been developed for determining rhein content, including thin layer chromatography [bib_ref] Analysis of anthraquinones in Rheum franzenbachii Münt (rhubarb) by thin-layer chromatography, Ma [/bib_ref] , high performance liquid chromatography (HPLC)-ultraviolet detection [bib_ref] Studies on the components of unprocessed and processed radixet rhizoma rhei, cortex..., Zhou [/bib_ref] , HPLC-mass spectrometry [bib_ref] An alternative solvent system for the separation of anthraquinone aglycones from rhubarb..., Danielsen [/bib_ref] , high performance capillary electrophoresis [bib_ref] Content determination of emodin and chrysophanol in sanhuangyou liniment by HPCE, Hu [/bib_ref] , and capillary electrophoresis chromatography . According to the provisions of the 2020 edition of the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2020), the total ash content of medicinal materials should not exceed 10.0% (General Rule 2302). The total anthraquinone content was determined using HPLC (General Rule 0512), with the total amount of aloe emodin (C 15 H 10 O 5 ), rhein (C 15 H 8 O 6 ), emodin (C 15 H 10 O 5 ), chrysophanol (C 15 H 10 O 4 ), and emodin methyl ether (C 16 H 12 O 5 ) not less than 1.5%. It shouldn't be less than 25.0% according to the hot-dip method, which is a water-soluble extract determination method (General Rule 2201). ## Pharmacological mechanisms of rhein on dm mellitus insulin resistance Insulin resistance (IR) is a common risk factor for various endocrine, metabolic, and cardiovascular diseases [bib_ref] Insulin resistance, cardiovascular stiffening and cardiovascular disease, Hill [/bib_ref]. Rhein can significantly improve IR associated with obesity [bib_ref] The anti-obesity effects of rhein on improving insulin resistance (IR) and blood..., Ji [/bib_ref] and non-alcoholic fatty liver disease [bib_ref] Rhein ameliorates fatty liver disease through negative energy balance, hepatic lipogenic regulation,..., Sheng [/bib_ref]. Similarly, rhein can play a role in reducing IR, thereby inhibiting DM progression. Insulin resistance prevents insulin from performing its normal physiological function, causing high blood glucose. This results in hyperinsulinism and a series of changes such as hyperglycemia and obesity. Rhein significantly improves glucose tolerance without regulating insulin secretion in streptozotocin (STZ)-induced diabetic mice [bib_ref] Insulin sensitizing and alpha-glucoamylase inhibitory action of sennosides, rheins and rhaponticin in..., Choi [/bib_ref]. It was found to significantly reduce blood glucose, glycosylated hemoglobin, and glycosylated serum protein levels, increase the insulin sensitivity index, and decrease the IR index, Homeostatic Model Assessment for Insulin Resistance, effectively improving insulin sensitivity in diabetic rats [bib_ref] Rhein improves insulin sensitivity of diabetic rats by increasing the protein expression..., Chi [/bib_ref]. These findings confirm that rhein enhances insulin sensitivity. The hexosamine pathway (HBP) is an intracellular glucose metabolism pathway. Under normal circumstances, only 1%-3% of glucose enters the HBP. Changes in intracellular HBP activity are associated with the development of IR [bib_ref] Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport..., Marshall [/bib_ref] [bib_ref] Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: Decreased..., Cooksey [/bib_ref]. Studies have shown that rhein inhibits the development of DM mainly by inhibiting the overactivity of HBP. Specifically, after rhein stimulation, the activity of glutamine fructose-6-phosphate aminotransferase (GFAT) in the first step of the HBP decreases, and the formation of UDP-N-acetylglucosamine, the final product of HBP, decreases significantly [bib_ref] High glucose-induced mesangial cells transforming growth factorβ1 synthesis is mediated by upregulated..., Liu [/bib_ref] [bib_ref] Glucose transporter in human glomerular mesangial cells modulated by transforming growth factor-beta..., Liu [/bib_ref]. Glucose transporter 1 (GLUT1) is the key glucose transporter in mesangial cells and is responsible for their basal metabolism; its expression level is often the main rate-limiting step in cellular glucose metabolism. Modern research has found that rhein can significantly reduce the glucose uptake rate of mesangial cells were transduced with the human GLUT1 gene (MCGT1) in a dosedependent manner, correct the hypertrophic state of MCGT1, and reduce the cell volume of MCGT1 and the ratio of RNA/DNA and protein/DNA [bib_ref] Glucose transporter in human glomerular mesangial cells modulated by transforming growth factor-beta..., Liu [/bib_ref] [bib_ref] Identification and function of glucose transporter 1 in human mesangial cells, Li [/bib_ref] [bib_ref] Inhibition of glucose transporter 1 overexpression in mesangial cells by rhein. Chin, Zhu [/bib_ref]. In mesangial cells, transforming growth factor-β (TGF-β1) stimulates extracellular matrix (ECM) synthesis and cell hypertrophy as a result of high glucose concentrations [bib_ref] Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: Implication of gangliosides, Masson [/bib_ref] [bib_ref] Hyperglycemia and glucosamine-induced mesangial cell cycle arrest and hypertrophy: Common or independent..., Masson [/bib_ref]. Rhein inhibits the overactivity of HBP in rat mesangial cells (MCGT1) transfected with the GLUT1 gene. It reduces the expression of TGF-β1, number of hypertrophic cells, and ECM synthesis [bib_ref] The effect of glucose transporter 1 on hexosamine biosynthesis pathway in rat..., Liu [/bib_ref] [bib_ref] Rhein reverses the diabetic phenotype of mesangial cells over-expressing the glucose transporter..., Zheng [/bib_ref]. Rhein inhibits the upregulation of plasminogen activator inhibitor-1 (PAI-1) mRNA expression in TGF-β1-induced endothelial cells in a dose-dependent manner and the production of endothelial PAI-1 protein and has a significant inhibitory effect on the activity of phosphor-p44/p42 mitogen-activated protein kinase (MAPK) induced by TGF-β1 in human endothelial cells [bib_ref] Rhein inhibits transforming growth factor beta1 induced plasminogen activator inhibitor-1 in endothelial..., Zhu [/bib_ref]. Rhein is transmitted through protein kinase C (PKC), and MAPK inhibits the abnormal increase in GLUT1 mRNA expression, thereby antagonizing the pathological effects of TGF-β1 on mesangial cells [bib_ref] Anti-cancer properties of anthraquinones from rhubarb, Huang [/bib_ref] [bib_ref] Rhein ameliorates lipopolysaccharide-induced intestinal barrier injury via modulation of Nrf2 and MAPKs, Zhuang [/bib_ref]. GFAT is the key enzyme in HBP, and its activity intensity reflects the active state of HBP. Liu Q. found that rhein inhibits the activity and expression of GFAT in the skeletal muscle of IR mice, 3T3 L1 adipocytes, and C2C12 myoblast models of IR induced by high glucose and insulin levels, thereby inhibiting HBP flow and downregulating the glycosylation modification level of the protein O-linked N-acetylglucosamine. It increases the responsiveness of the whole body and insulin target tissues to insulin. The pharmacological mechanisms by which rhein improves IR in DM by inhibiting HBP overactivity are shown in [fig_ref] FIGURE 3: The pharmacological mechanisms of rhein in improving IR in DM by inhibiting... [/fig_ref]. In addition, rhein can increase the expression of SIRT1 in the renal tissue, improve insulin sensitivity and glucose and lipid metabolism disorders, and reduce the IR index in diabetic rats . A study found that argirein synthesized by hydrogen bonding of rhein and L-arginine inhibits the activation of NOX4-dependent O 2 − in rat aortic endothelial cells triggered by palmitic acid, thereby inhibiting endothelial IR and improving vascular function . Rhein significantly reduces blood glucose levels in diabetic mice and upregulates peroxisome proliferator-activated receptor γ (PPARγ) expression, thus improving metabolic disorders and reversing IR [bib_ref] Rhein improves insulin sensitivity of diabetic rats by increasing the protein expression..., Chi [/bib_ref]. ## Inflammation The risk of developing DM is positively associated with inflammation. Rhein exhibits significant anti-inflammatory properties in colitis [bib_ref] Anti-inflammatory effect of Rhein on ulcerative colitis via inhibiting PI3K/Akt/mTOR signaling pathway..., Dong [/bib_ref] [bib_ref] Rhein regulates redox-mediated activation of NLRP3 inflammasomes in intestinal inflammation through macrophage-activated..., Zhou [/bib_ref] , acute pancreatitis , and obesity [bib_ref] Rhubarb hydroxyanthraquinones act as antiobesity agents to inhibit adipogenesis and enhance lipolysis, Fang [/bib_ref] , among other conditions. Similarly, rhein exerts anti-inflammatory effects by inhibiting DM development. Inflammation induces the release of chemicals called inflammatory cytokines from damaged cells; these cytokines regulate various inflammatory responses. Studies have shown that rhein inhibits the development of DM primarily by inhibiting the release of inflammatory cytokines. In the KK/HlJ diabetic mouse model, rhein lysinate reduces the levels of blood glucose and significantly decreases the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) [bib_ref] Rhein lysinate decreases inflammation and adipose infiltration in KK/HlJ diabetic mice with..., Wei [/bib_ref]. Another study reported that Rhein@OR-S gel can inhibit reactive oxygen species (ROS) and NO overproduction and has excellent efficiency in suppressing diabetic wound inflammation, bringing forward the transition from the inflammatory phase to the proliferative phase [bib_ref] Self-assembled herbal medicine encapsulated by an oxidation-sensitive supramolecular hydrogel for chronic wound..., Zhao [/bib_ref]. Nuclear factor-kappa B (NF-κB) is a sequence-specific DNAbinding protein with pleiotropic transcriptional regulation, regulating the expression of multiple target genes encoding cytokines and inflammatory mediators. The NF-κB signaling pathway plays an important role in the molecular mechanisms of The pharmacological mechanisms of rhein in improving IR in DM by inhibiting HBP overactivity. PAGM, Phosphoacetllucosaminemutase; ATP, adenosine triphosphate; ADP, adenosine diphosphate; UDP, uridine diphosphate; UTP, uridine triphosphate. Frontiers in Pharmacology frontiersin.org IR. After stimulation by exogenous substances, such as TNF-α, IL-1β, and LPS, IκB-α in the cytoplasm is phosphorylated, undergoes ubiquitination and degradation, and is released into p65 into the nucleus. In turn, p65 binds to specific response element sequences on target genes and activates their transcription [bib_ref] NF-kappaB: Two sides of the same coin, Pires [/bib_ref]. Rhein plays an anti-inflammatory role by inhibiting the NF-κB signaling pathway through the degradation of IκB-α and nuclear translocation of p65 [bib_ref] NF-kappaB regulation in the immune system, Li [/bib_ref] [bib_ref] Diacerhein improves glucose tolerance and insulin sensitivity in mice on a high-fat..., Tobar [/bib_ref] [bib_ref] Directed self-assembly of herbal small molecules into sustained release hydrogels for treating..., Zheng [/bib_ref] [bib_ref] Diacerein attenuates vascular dysfunction by reducing inflammatory response and insulin resistance in..., He [/bib_ref]. Macrophages exacerbate inflammatory responses by releasing inflammatory factors. Rhein plays a dual role in lipopolysaccharide-activated macrophages by inhibiting IKKβ. On the one hand, by inhibiting the standard IKKβ-mediated NF-κB activation sequence (IKKβ axis), rhein suppresses downstream NO and IL-6 levels; on the other hand, rhein enhances caspase-1 activity by inhibiting NF-κB-independent IKKβ itself, which in turn increases the release of IL-1β and HMGB1. PPARγ is a significant protein involved in insulin activity. PPAR-γ ligand downregulates the expression of TNF-α in macrophages PPAR-γ [bib_ref] Leukocyte overexpression of intracellular NAMPT attenuates atherosclerosis by regulating pparγ-dependent monocyte differentiation..., Bermudez [/bib_ref]. C-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family, which is a critical component of the death pathway caused by inflammation and oxidative stress. Rhein can regulate the PPARγ signaling pathway in a dose-dependent manner, effectively improving the protein expression of PPARγ, and inhibiting NF-κB expression and p-JNK activation. These changes effectively inhibit the inflammatory response of renal mesangial cells and enhance insulin sensitivity [bib_ref] Effect of rheic acid on inflammatory cytokines in rats with diabetic nephropathy, Duan [/bib_ref] [bib_ref] The anti-obesity effects of rhein on improving insulin resistance (IR) and blood..., Ji [/bib_ref]. The Wnt/β-catenin signaling pathway activated by high glucose levels is involved in the pathogenesis of DM [bib_ref] Identification of a novel inhibitor of the canonical Wnt pathway, Park [/bib_ref] [bib_ref] A new link between diabetes and cancer: Enhanced WNT/β-catenin signaling by high..., García-Jiménez [/bib_ref]. ß-Catenin is mainly responsible for mediating intercellular adhesion and related signaling. Stimulation of the Wnt signaling pathway is the only mechanism that enhances ß-catenin stability [bib_ref] Beta-catenin controls hair follicle morphogenesis and stem cell differentiation, Lin [/bib_ref]. When the Wnt signaling pathway is activated, the Wnt protein binds to the receptor, thereby inhibiting the activity of GSK-3β. Thus, ß-catenin cannot be phosphorylated and degraded, then accumulates in the cytoplasm, and integrates into the nucleus to initiate the expression of downstream target genes. Rhein can downregulate the expression of ß-catenin protein, promote the expression of GSK-3β, reduce the secretion of TGF-β1, and play a role in inhibit the Wnt/β-catenin signaling pathway . The NLRP3 inflammasome is involved in inflammation and glucose homeostasis, and its activation can induce immune cells to produce proinflammatory cytokines, thereby exacerbating IR. NLRP3 inflammasome provides a platform for the production of IL-1β. IL-1β directly inhibits the insulin signaling pathway by decreasing insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and negatively regulating IRS-1 gene expression [bib_ref] Modulatory mechanisms of the NLRP3 inflammasomes in diabetes, Ding [/bib_ref]. Rhein inhibits NLRP3 inflammasome activation [bib_ref] Rhein attenuates inflammation through inhibition of NF-κB and NALP3 inflammasome in vivo..., Ge [/bib_ref]. Rhein analogs may also improve diabetic symptoms by reducing the production of pro-inflammatory cytokines and inhibiting the expression of TGF-β1. Rhein lysinate (RHL), a water-soluble rhein derivative, can reduce the expression of TNF-α and NF-κB, and inhibits the immune response by directly or indirectly blocking the TNF-NF-κB biochemical pathway [bib_ref] The protection of Rhein lysinate to liver in diabetic mice induced by..., Lin [/bib_ref] [bib_ref] Rhein lysinate protects renal function in diabetic nephropathy of KK/HlJ mice, Lin [/bib_ref] [bib_ref] Diacerein alleviates kidney injury through attenuating inflammation and oxidative stress in obese..., Chueakula [/bib_ref]. Diacerhein is completely metabolized in the human body, resulting in rhein production, which exerts antiinflammatory properties locally, decreases the expression of proinflammatory cytokines such as IL-1β, TNF-α, IFN-γ, and IL-12, thereby interfering with the occurrence of DM [bib_ref] Diacerhein downregulate proinflammatory cytokines expression and decrease the autoimmune diabetes frequency in..., Malaguti [/bib_ref]. Diacerein can reduce the level of TNF-α in patients with DM and improve metabolic control [bib_ref] Effect of diacerein on renal function and inflammatory cytokines in participants with..., Piovesan [/bib_ref] [bib_ref] Effect of diacerein on metabolic control and inflammatory markers in patients with..., Tres [/bib_ref]. The experimental details of the anti-inflammatory mechanisms of rhein are presented in [fig_ref] TABLE 1: Experiments on the anti-inflammatory mechanism of rhein in diabetes mellitus [/fig_ref]. ## Oxidative stress Under high glucose conditions, autoxidation of glucose, nonenzymatic glycosylation of glucose and other biomolecules, and enhancement of polyol metabolic pathways results in the production of large amounts of ROS in the body. The inhibition of antioxidants (vitamin C, vitamin E, etc.,) and antioxidant enzymes (SOD, glutathione (GSH, catalase, etc.,) used to scavenge ROS in the body leads to the accumulation of lipid peroxidation products (e.g., MDA) and a long-term imbalance between the production of highly reactive molecules and antioxidant effects, resulting in oxidative stress, which in turn activates the polyol, AGEs, and PKC pathways, causing IR and impairment of islet ß-cell function [bib_ref] Oxidative stress and antioxidant treatment in diabetes, Scott [/bib_ref]. Rhein has a unique structure containing carboxyl and hydroxyl groups, including two hydroxyl radicals, which confer antioxidant properties. Rhein inhibits OS in histiocytes by inhibiting antioxidant enzymes and reducing coenzyme II (NADPH) oxidase, reducing peroxisomes such as ROS in vivo, and has a protective effect on islet function and renal histiocytes. Huang et al. applied rhein to treat STZ-induced DM rats and found that rhein could antagonize the effect of Ang II and inhibit the signaling pathway of Ang II receptor, significantly inhibit the mRNA expression of renal NADPH oxidase subunits p47 phox and p22 phox . This reduces the production of ROS, which increases the antioxidant capacity of kidney tissue in DM and inhibits the oxidative emergency of kidney tissue [bib_ref] Effect of rhein on NADPH gene expression in the kidney of diabetic..., Huang [/bib_ref]. reported that rhein could reduce the expression of 8-OHdG, a marker of DNA damage, significantly inhibit OS injury in islet cells, and protect islet function [bib_ref] Effects of rheinic acid on markers of insulin secretion, inflammation and oxidative..., Huang [/bib_ref]. Rhein inhibited the increase in MDA content, increased SOD activity, and significantly inhibited OS damage in pancreatic islet cells [bib_ref] Toxic effects caused by rhubarb (Rheum palmatum L.) are reversed on immature..., Wang [/bib_ref] [bib_ref] Protective effects of lysine rhein on kidney of diabetic mice, Li [/bib_ref]. The pharmacological mechanism of rhein in inhibition of oxidative stress in DM are shown in . ## Gut microbiota Gut microbiota dysbiosis leads to an increase in the proportion of G-bacteria, which, through the production and uptake of more LPS, activates a low-grade chronic inflammatory response, exacerbates IR, and impairs ß-cell function [bib_ref] Gut microbiota in human adults with type 2 diabetes differs from non-diabetic..., Larsen [/bib_ref]. A two-stage metagenome-wide association study based on deep next-generation shotgun sequencing of gut microbial DNA extracted from 345 individuals identified alterations in vitamin and cofactor synthesis-related, butyrate-producing, sulfate-reducing, and mucindegrading bacteria in the gut of patients with DM. The gut environment of patients with DM is one that stimulates bacterial defense mechanisms against OS [bib_ref] A metagenome-wide association study of gut microbiota in type 2 diabetes, Qin [/bib_ref]. The ratio of Bacteroidetes to Firmicutes was positively correlated with blood glucose concentration. Rhein significantly enriched the gut microbiota, increased the relative abundance of Bacteroides, Frontiers in Pharmacology frontiersin.org reversed the ratio of Bacteroides and Firmicutes, maintained the diversity of the gut microbiota to improve glucose metabolism, and exerted a hypoglycemic effect . Rhein significantly increased the number of terminal ileal L cells in db/db mice, which increased glucagon-like tide-l (GLP-1) release, which in turn stimulated increased insulin secretion by islet ß-cells and inhibited glucagon secretion by α-cells, thus improving glucose homeostasis . ## Dyslipidemia Diabetic dyslipidemia, which comprises very-low-density lipoprotein (VLDL), triacylglycerol (TG), small dense low-density lipoprotein, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), is common in patients with DM [bib_ref] Mechanisms and treatment of dyslipidemia in diabetes, Bahiru [/bib_ref]. Multiple polymorphic studies have confirmed that hyperglycemia and elevated plasma triglycerides are associated with hepatic steatosis [bib_ref] The CDKAL1 gene is associated with impaired insulin secretion and glucose-related traits:..., Liang [/bib_ref]. 3-hydroxy-3methyl glutaryl coenzyme A (HMG-CoA), a rate-limiting enzyme in cholesterol synthesis by hepatocytes, catalyzes the formation of mevalonate. Gao et al. found that rhein inhibits HMG-CoA reductase, thereby inhibiting cholesterol synthesis [bib_ref] Rhein improves renal lesion and ameliorates dyslipidemia in db/db mice with diabetic..., Gao [/bib_ref]. Wu et al. found that rhein significantly reduced the contents of TG and TC in L02 hepatic steatosis cells, and significantly reduced the fat granules around the nucleus [bib_ref] Rapid screening of active components from Rhei Radix et Rhizoma for hypolipidemia..., Wu [/bib_ref]. Rhein can downregulate the expression of resistin in the adipose tissue of obese diabetic rats, thereby reducing plasma free fatty acid levels and inhibiting signal transduction to insulin . Under pathological conditions such as DM and fatty liver, oxidized lowdensity lipoprotein can induce an increase in PPAR-γ through the PKC-α/ERK/PPAR-γ pathway [bib_ref] Nonalcoholic steatohepatitis: Summary of an AASLD single topic conference, Neuschwander-Tetri [/bib_ref] , which can inhibit the susceptibility of PPAR-γ and the expression of its target genes in a dose-dependent manner [bib_ref] Protective effects of rhein on nonalcoholic fatty liver disease in rats fed..., Cen [/bib_ref]. Rhein can reduce serum total cholesterol (TC) levels, increase HDL levels, reduce LDL and VLDL levels, and prevent excessive oxidation of LDL in mice [bib_ref] Study on the extraction process of total anthraquinones in Radix et Rhizoma..., Xie [/bib_ref]. ## Mitochondrial dysfunction When the inner and outer mitochondrial membrane proton gradients are high and oxygen consumption is low, ROS generation in the body increases. Excessive ROS damage mitochondrial proteins, mtDNA, and lipids on the mitochondrial membrane, thereby causing mitochondrial damage. Sustained and high-yield ROS generation caused by mitochondrial function damage reduces ATP generation, damages ß cells and inhibits insulin release [bib_ref] Identification of cross-species shared transcriptional networks of diabetic nephropathy in human and..., Hodgin [/bib_ref]. ROS prevents IRS-1 from binding to the insulin receptor by activating IKKβ, which phosphorylates the IRS-1 SER site, thereby preventing the transmission of insulin signals and causing insulin resistance [bib_ref] Impact of mitochondrial ROS production in the pathogenesis of insulin resistance, Nishikawa [/bib_ref]. Rhein is localized in the mitochondria of pancreatic ß-cells, and it can maintain mitochondrial ultrastructure by reducing the ROS content in mitochondria, thereby inhibiting the expression of dynamin related protein 1 (Drp1) and preventing ß-cell apoptosis induced by hyperglycemia [bib_ref] Rhein induces apoptosis through induction of endoplasmic reticulum stress and Ca2+-dependent mitochondrial..., Lin [/bib_ref] [bib_ref] Rhein protects pancreatic β-cells from dynamin-related protein-1-mediated mitochondrial fission and cell apoptosis..., Liu [/bib_ref]. AMP-activated protein kinase (AMPK) is an important protein that regulates mitochondrial biosynthesis [bib_ref] AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of..., Jager [/bib_ref]. Studies have shown that rhein can activate the AMPK-Sirt1 pathway to promote the translocation of the α-subunit of ATPase to the plasma membrane, promote mitochondrial biosynthesis, improve mitochondrial respiratory function, and ultimately improve insulin resistance [bib_ref] Rhein inhibits autophagy in rat renal tubular cells by regulation of AMPK/mTOR..., Tu [/bib_ref]. ## Β-cell failure Insufficient relative or absolute insulin secretion due to an insufficient number of pancreatic ß-cells causes hyperglycemia [bib_ref] beta-cell failure in diabetes and preservation by clinical treatment, Wajchenberg [/bib_ref]. Type 2 DM is the result of progressive loss of pancreatic ß-cell number and secretion function [bib_ref] Increase in insulin secretion induced by panax ginseng berry extracts contributes to..., Park [/bib_ref]. The main mechanism for the decrease in ß-cell numbers is an increase in ß-cell apoptosis rather than a decrease in proliferation or replication [bib_ref] Beta-cell apoptosis in type 2 diabetes: Quantitative and functional consequences, Lupi [/bib_ref]. O 2 treatment to NIT-1 cells as a model of pancreatic cell damage and found that RHL increased the expression level of anti-apoptotic protein Bcl-2, decreased the expression level of pro-apoptotic protein Bax, and increased the Bcl-2/Bax ratio, which partially blocked NIT-1 cell apoptosis induced by H 2 O 2 and protected pancreatic ß cells, thus exerting a pancreatic protective effect [bib_ref] Rhein lysinate protects pancreas in type 2 diabetic mice, Hu [/bib_ref]. Rhein significantly inhibited the reduction of ß-cell mass and reduced ßcell apoptosis, which improved the glucose-dependent and glucoseindependent secretion of insulin by improving ß-cell function [bib_ref] Improvement of glucose tolerance by rhein with restored early-phase insulin secretion in..., Du [/bib_ref] [bib_ref] Rhein protects pancreatic β-cells from dynamin-related protein-1-mediated mitochondrial fission and cell apoptosis..., Liu [/bib_ref]. The pharmacological mechanisms of rhein in DM are presented in [fig_ref] FIGURE 5: Pharmacological mechanisms of rhein in diabetes mellitus [/fig_ref]. ## Toxicology of rhein In recent years, increasing adverse reactions due to high doses or long-term administration of rhubarb and its preparations have caused global concern [bib_ref] The protective and toxic effects of rhubarb tannins and anthraquinones in treating..., Zeng [/bib_ref] [bib_ref] In vitro study of the nephrotoxicity of total Dahuang (Radix Et Rhizoma..., Cao [/bib_ref]. Therefore, it is crucial to determine its toxic components. Studies have shown that rhein exhibits hepatotoxicity and nephrotoxicity [bib_ref] Hepatotoxicity and Nephrotoxicity of Rhei Radix et Rhizoma and Its Attenuation Methods, Hu [/bib_ref] [bib_ref] Rhein protects against severe acute pancreatitis in vitro and in vivo by..., Yang [/bib_ref]. Rhein can inhibit HepaRG cell viability and induce cell death by blocking the S-phase cell cycle and activating the Fas-and mitochondria-mediated apoptotic pathways [bib_ref] Rhein induces cell death in HepaRG cells through cell cycle arrest and..., You [/bib_ref]. Rhein affects the accumulation of Rh123 in hepatocyte mitochondria, thereby altering the mitochondrial membrane potential and affecting mitochondrial membrane permeability; mitochondrial dysfunction causes changes in Ca 2+ homeostasis and ATP depletion, ultimately leading to cell death [bib_ref] The hepatotoxicity of rhein involves impairment of mitochondrial functions, Bironaite [/bib_ref]. He et al. found that CYP2C19 could activate rhein to reactive metabolites (RMs), and RMs can covalently bind to intracellular mitochondria, resulting in ## Figure 4 Pharmacological mechanism of rhein in inhibition of oxidative stress in diabetes mellitus. Drpl, dynamin-relatedprotein 1; Nrf2, nuclea factor erythroid-2-related factor 2; MDA,malonaldehyde; ROS, reactive oxygen species; SOD,superoxide dismutase; HO-1, Heme Oxygenase-1; CAT,catalase; GPX, glutathione peroxidase; GSH, glutathione,reduced; GSSG glutathione,oxydized; GR, gluathione reductase; NADPH, nicotinamide adenine dinucleotide phosphate; G-6-PDH,glucose-6-phosphate dehydrogenase; G-6-P, glucose-6-phosphate; GPI, phosphohexose isomerase; F-6-P, fructose-6phosphate; PEK, Phosphofructokinase; PGAL, glyceraldehyde-3-phosphate; 1,3 BPG, 1,3 Biphosphoglycerate; TCA cycle, tricarboxylicacidcycle. Frontiers in Pharmacology frontiersin.org ROS overproduction, respiratory chain dysfunction, liver function impairment (increased aspartate aminotransferase and lactate dehydrogenase), and cell death. In addition, RM overproduction depletes GSH, leading to hepatotoxicity. Mao et al. demonstrated that rhein can induce apoptosis in HK-2 cells by activating caspase-3 and caspase-9, which are involved in the ROSdependent mitochondrial pathway [bib_ref] The UCP2-related mitochondrial pathway participates in rhein-induced apoptosis in HK-2 cells, Mao [/bib_ref]. Rhein induces apoptosis by activating caspase-3 expression, resulting in pathological changes in renal histology, mainly manifested as protein casts in the lumen of renal tubules, capillary congestion in glomeruli and renal interstitium, swelling of renal tubule epithelial cells, and small focal proliferation of lymphocytes [bib_ref] Hepatotoxicity and Nephrotoxicity of Rhei Radix et Rhizoma and Its Attenuation Methods, Hu [/bib_ref]. Repeated administration of rhein for 75 days resulted in obvious steatosis and cell swelling in the liver, as well as varying degrees of inflammation, swelling, and necrosis in the kidney and colon cells, associated with competitive binding of rhein to multidrug resistanceassociated protein 2 (MRP2) protein sites. Reduced expression of MRP2 protein blocked cellular oxidative stress substance expulsion, causing increased oxidative stress, impaired mitochondrial function, and stimulation of the release of caspase 3, a downstream factor of apoptosis, thus exhibiting cytotoxicity. ## Pharmacokinetics of rhein The HPLC-fluorescence detector method has been used to study the absorption of rhein in rats after oral administration, and the results showed that rhein is mainly distributed in the stomach, kidney, liver, and intestine, had good biocompatibility with them, and could pass the blood-brain barrier. Rhein is mainly metabolized by the kidneys. The cumulative excretion rate in urine is 6.0 ± .6% within 24 h after oral administration of 70 mg/kg rhein, and the renal clearance of rhein is 21.3 ± 5.47 mL/h/kg [bib_ref] Prediction of human pharmacokinetics from preclinical information of rhein, an antidiabetic nephropathy..., Hao [/bib_ref]. In total, 20% of rhein is excreted in its natural form in urine, 60% as thiourea acid ester conjugates, and 20% as sulfates. Rhein is the only anthraquinone component of rhubarb that is absorbed into the blood in the human body [bib_ref] Pharmacokinetic analysis of rhein in Rheum undulatum L, Lee [/bib_ref]. detection to determine the plasma levels of rhein, which rapidly reached the maximum level after oral administration. In addition, the plasma concentration-time curve revealed secondary peaks, indicating enterohepatic recirculation of rhein.used liquid chromatograph mass spectrometer to compare the pharmacokinetics of rhein in rat plasma after oral administration of rhubarb peony decoction (RPD) and rhubarb extract and found that the C max of rhein in RPD was significantly lower than that after administration of rhubarb, indicating that the absorption of rhein in rats was inhibited after oral administration of RPD. [bib_ref] Determination of bioactive components in Chinese herbal formulae and pharmacokinetics of rhein..., Hou [/bib_ref] applied Ultra-high performance liquid chromatography-tandem mass spectrometry to simultaneously quantify the active ingredients in commercial ready-to-use pharmaceutical herbal products to investigate the pharmacokinetics of rhein, rhubarb, and San-Huang-Xie-Xin-Tang (SHXXT) in rats and found that C max and area under the curve (AUC) in the SHXXT group, compared with the rhein administration group, were significantly increased by 17-fold and 9-fold, respectively, indicating that the absorption rate of rhein via SHXXT was significantly higher than that of pure rhein. Based on the pharmacokinetic parameters of rhein, herbal formulations containing different components may have a synergistic or antagonistic effect on rhein absorption. Considering its hepatotoxicity and nephrotoxicity, it Pharmacological mechanisms of rhein in diabetes mellitus. Rhein maintains glucose homeostasis by anti-inflammation, alleviating renal lesions, improving insulin resistance, improving lipid metabolism, anti-oxidative stress, maintaining mitochondrial respiratory function, improving gut microbiota, and improving β -cell functional. GSK-3 β, Glycogen synthase kinase 3 β; PPAR-γ ,peroxisome proliferator-activated receptor γ ; NF-κ B, nuclear factor kappa-B; HDAC3, histone deacetylase 3; NLRP3, NOD-like receptor thermal protein domain associated protein 3; IL-6, Interleukin-6; IL-1 β ,Interleukin-1 β ; TNFα ,tumor necrosis factor α ; IKK β ,inhibitor of kappa B kinase β ;Foxo3 α, Forkhead box 03 α ; SIRT1, sirtuin 1; HBP, Hexosamine pathway; TC, Serum total cholesterol; HDL-C,High density liptein cholesterol; TG, triacylglycerol; LDL-C, Low-Density Lipoprotein Cholesterol; VLDL,very low-density lipoprotein; SOD, Superoxide dismutase; CAT,Catalase from micrococcus lysodeiktic; GSH,glutathione,reduced; GSSG, glutathione, oxydized; MDA, malonaldehyde; ROS, reactive oxygen species; Drpl,Dynamin-related protein 1; AMPK,Adenosine 5' -monophosphate (AMP)-activated protein kinase; GLP-1,glucagon-like peptide-1; Bcl-2,B-cell lymphoma-2; BAX,BCL2-Associated X. Frontiers in Pharmacology frontiersin.org is crucial to ensure that herbs rich in rhein are used in the appropriate formulations in TCM. Researchers have begun to improve the absorption efficiency and bioavailability of rhein by altering the drug delivery route, using liposomes, nanoparticles, solid lipid nanoparticles, polymeric micelles, and hydrogels, among others [bib_ref] A research update on the therapeutic potential of rhein and its derivatives, Cheng [/bib_ref] [bib_ref] Aramid nanofibersreinforced rhein fibrous hydrogels as antibacterial and anti-inflammatory burn wound dressings, Li [/bib_ref]. F127 modified liposomal rhein (F127-RPC-Lip) exhibits longer systemic circulation time, better drug distribution, and reduced pancreatic injury after complexation with lipids via non-covalent interactions. an emulsification solvent evaporation technique to encapsulate rhein, which has poor water solubility, in mPEG-PLA NPs. RH-NPs significantly increase the plasma AUC of rhein, improve the biological half-life of rhein, increase the circulation time of rhein, and enhance renal permeability . (1.5%), Precrol ATO5 (2%), and lecinol (.5%) were prepared by hot homogenization followed by ultrasonication after co-solubilization in a mixture of organic solvent consisting of acetone/ethanol (1:1) to Rhein-SLNs. The AUC 0-t of rhein in the case of Rhein-SLNs was 2.06 times higher than that of the suspension group [bib_ref] Preparation, characterization, and in vivo study of rhein solid lipid nanoparticles for..., Feng [/bib_ref]. DOX/RHE-loaded polymeric micelle, assembled by DSPE -PEG2000 and TPGS1000, released rhein in a rapid and targeted manner until 10 h to increase the distribution of rhein in tumor tissues [bib_ref] Doxorubicin and rhein loaded nanomicelles attenuates multidrug resistance in human ovarian cancer, Han [/bib_ref]. Rhein hydrogel, directly self-assembled from rhein via intermolecular π-π interactions and hydrogen bonds, readily binds to Toll-like receptor four and then significantly dephosphorylates IκBα to inhibit the nuclear translocation of p65 in the NF-κB signaling pathway [bib_ref] Directed self-assembly of herbal small molecules into sustained release hydrogels for treating..., Zheng [/bib_ref]. These new delivery systems improve the stability, absorption efficiency, and bioavailability of rhein. ## Concluding remarks The effects of rhein on DM have been studied extensively. Rhein ameliorates IR by inhibiting the overactivity of HBP and the production of inflammatory cytokines through signaling pathways such as those of NF-κB and Wnt/β-catenin. Rhein inhibits ROS overproduction, thereby preventing oxidative stress in cells. It maintains the diversity of the gut microbiota to improve glucose metabolism. Moreover, it improves lipid metabolism, mitochondrial respiratory function, and pancreatic ß-cell function. The toxicology and pharmacokinetics of rhein have been studied in detail. In view of the low bioavailability and solubility of rhein, further structural modification studies can be conducted to search for derivatives with low toxicity and better efficacy that can be better applied in the treatment of DM. # Author contributions TD and JH: the conception and design of the study. MY: the conception and design of the study and drafting the article. JD: drafting the article. BC, ZW and ZZ: revising the article critically. YY and MY: revising the article. All authors contributed to the article and approved the submitted version. # Funding This study was financially supported by the Natural Science [fig] FIGURE 1: Plant and Slice of root of Rheum palmatum L. The leaves of Rheum palmatum L. are palmately lobed to half-lobed, with the lobes mostly narrowly triangular; its flowers are small, purple-red or yellowish-white; its buds are inverted pyramidal; and its fruits are small. The outer skin of rhubarb is yellowishbrown or reddish-brown, with white-like reticulate texture and scattered stars, and its broken surface is light reddish-brown or yellowish-brown, showing granularity; it has a fresh aroma and a bitter and slightly astringent taste. (A) Rheum palmatum L. is a kind of herb with medicinal value. (B) prepared slices of Rhubarb, containing a variety of active pharmacological ingredients. [/fig] [fig] FIGURE 3: The pharmacological mechanisms of rhein in improving IR in DM by inhibiting HBP overactivity. PAGM, Phosphoacetllucosaminemutase; ATP, adenosine triphosphate; ADP, adenosine diphosphate; UDP, uridine diphosphate; UTP, uridine triphosphate. [/fig] [fig] FIGURE 5: Pharmacological mechanisms of rhein in diabetes mellitus. Rhein maintains glucose homeostasis by anti-inflammation, alleviating renal lesions, improving insulin resistance, improving lipid metabolism, anti-oxidative stress, maintaining mitochondrial respiratory function, improving gut microbiota, and improving β -cell functional. GSK-3 β, Glycogen synthase kinase 3 β; PPAR-γ ,peroxisome proliferator-activated receptor γ ; NF-κ B, nuclear factor kappa-B; HDAC3, histone deacetylase 3; NLRP3, NOD-like receptor thermal protein domain associated protein 3; IL-6, Interleukin-6; IL-1 β ,Interleukin-1 β ; TNFα ,tumor necrosis factor α ; IKK β ,inhibitor of kappa B kinase β ;Foxo3 α, Forkhead box 03 α ; SIRT1, sirtuin 1; HBP, Hexosamine pathway; TC, Serum total cholesterol; HDL-C,High density liptein cholesterol; TG, triacylglycerol; LDL-C, Low-Density Lipoprotein Cholesterol; VLDL,very low-density lipoprotein; SOD, Superoxide dismutase; CAT,Catalase from micrococcus lysodeiktic; GSH,glutathione,reduced; GSSG, glutathione, oxydized; MDA, malonaldehyde; ROS, reactive oxygen species; Drpl,Dynamin-related protein 1; AMPK,Adenosine 5' -monophosphate (AMP)-activated protein kinase; GLP-1,glucagon-like peptide-1; Bcl-2,B-cell lymphoma-2; BAX,BCL2-Associated X. [/fig] [table] TABLE 1: Experiments on the anti-inflammatory mechanism of rhein in diabetes mellitus. [/table]

The roles of highly conserved, non‐catalytic residues in class A β‐lactamases S2Supplementary Figure 1. Gels displaying soluble fractions of cell cultures expressing WT and mutant BlaC. NCnegative control, cells containing empty vector. Red frame shows the position of BlaC at 31 kDa, green frame shows the position of E. coli protein that was used for signal scaling. Non-labeled lanes contain mutants of residues with lower percent conservation, thus not discussed within this study.SupplementaryFigure 3. Nitrocefin kinetic curves. Error bars represent the standard deviation of a triplicate measurement. Lines represent the Michaelis-Menten fit.S5 Supplementary Figure 4. Negative derivative of the RFU signal from thermal shift assay for WT and mutant BlaC.S6 Supplementary Figure 5. Examples of NMR spectra of cell lysates. The upper panel displays overlay of purified wild type in phosphate buffer pH 6.4 (in blue) and the soluble fraction of cell lysate of a culture expressing the wild type blaC gene (in black). The middle and bottom panels display the overlays of the cell lysate spectra of the secondshell mutant D245H (in orange) with wild type BlaC (in black) and of the third-shell mutant A223K (in purple) with wild type BlaC (in black).S7 Supplementary Figure 6. Examples of NMR spectra of cell lysates with unfolded protein. The panels display the overlays of spectra of the soluble fraction of cell lysates of mutants D131N (in pink, top), D179Q (in brown, middle) and N245Q (in turquoise, bottom) with that of wild type BlaC (in black). Detection of BlaC location after overexpression with and without a signal peptide for membrane localization. Most protein is found in membrane in construct with the signal peptide, which is shown on gel (left) and by the lighter color of the cells (right). Most protein is found in cytoplasm after expression in construct without the signal peptide with some protein detected in the membrane fraction, which can be possibly attributed to inclusion bodies formation due to overexpression (membrane fraction and inclusion bodies were not separated). ## S-10 - [fig] S3 Supplementary, Figure 2: Example of CD spectra acquired for negative control (NC) and wild type (WT) (left panel); spectra of wild type, a poorly produced mutant (L81M) and a folded mutant (T71S) after background correction (right panel). [/fig] [fig] S8 Supplementary, Figure 7: Example of the location of nuclei with significant CSP in a second-shell mutant (G217S) located on the protein surface. The mutated residue is shown in magenta sticks.Supplementary Figure 8. [/fig]

Phase Diagram for a Lysyl‐Phosphatidylglycerol Analogue in Biomimetic Mixed Monolayers with Phosphatidylglycerol: Insights into the Tunable Properties of Bacterial Membranes Ion pairing between the major phospholipids of the Staphylococcus aureus plasma membrane (phosphatidylglycerol -PG and lysyl-phosphatidylglycerol -LPG) confers resistance to antimicrobial peptides and other antibiotics. We developed 3adLPG, a stable synthetic analogue which can substitute for the highy-labile native LPG, in biophysical experiments examining the membrane-protecting role of lipid ion pairing, in S. aureus and other important bacteria. Here we examine the surface charge and lipid packing characteristics of synthetic biomimetic mixtures of DPPG and DP3adLPG in Langmuir monolayers, using a combination of complementary surfaceprobing techniques such as infrared reflection-absorption spectroscopy and grazing-incidence x-ray diffraction. The resultant phase diagram for the ion paired lipids sheds light on the mixing behavior of lipids in monolayer models of resistant phenotype bacterial membranes, and provides a platform for future biophysical studies.The aminoacyl lipids produced by a wide range of bacteria are becoming increasingly recognized as clinically relevant virulence factors, due to the role they play in phenotypic adaptations to the physical and biochemical stressors which confer intrinsic defence against infection.[1,2]The most widely studied of these lipids is lysyl-phosphatidylglycerol (LPG), for which the genomic regulation and biosynthetic pathways in Staphylococcus aureus, have recently been elucidated in some detail.[3,4]Data from microbiological assays, has shown that an increased proportion of LPG in S. aureus membranes correlates with resistance to both host defensive peptides[5]and membrane-active therapeutic antibiotics.[6]The mechanisms facilitating such resistances are assumed to involve the tuning of target membrane physical properties, notably those of interfacial charge and lipid ordering, which are influenced by the LPG content in the bacteria membranes.[7]Biophysical investigations into these phenomena have been hampered by the labile nature of native LPG, which is readily hydrolysed under mild conditions, therefore exposing any such study to the risk of artefact.[8,9]To this end, stable LPG analogues have been synthesized. One such analogue, lysyl-phosphatidylethanolamine (LPE) exhibited an inhibitory effect on antimicrobial peptide activity when incorporated into vesicles containing phosphatidylglycerol (PG).[10]A second analogue, 3-aza-dehydroxy lysyl-phosphatidylglycerol (3adLPG), facilitated enhanced membrane ordering and antimicrobial peptide resistance when mixed with PG under mildly acidic conditions,[11]which are known to promote PG/LPG ion pair formation in model bacterial membranes.[12]In order to gain a better understanding of the influence of lipid ion pairing on membrane structure and interfacial properties, we conducted a high-resolution study of biomimetic mixtures of dipalmitoyl-3adLPG and DPPG in Langmuir monolayers.The natural phospholipid composition (Figure 1A) observed in different S. aureus strains is dominated by PG ( � 40-70 %) and LPG ( � 30-55 %) with saturated and iso-branched fatty [a] T. Ion pairing between the major phospholipids of the Staphylococcus aureus plasma membrane (phosphatidylglycerol -PG and lysyl-phosphatidylglycerol -LPG) confers resistance to antimicrobial peptides and other antibiotics. We developed 3adLPG, a stable synthetic analogue which can substitute for the highy-labile native LPG, in biophysical experiments examining the membrane-protecting role of lipid ion pairing, in S. aureus and other important bacteria. Here we examine the surface charge and lipid packing characteristics of synthetic biomimetic mixtures of DPPG and DP3adLPG in Langmuir monolayers, using a combination of complementary surfaceprobing techniques such as infrared reflection-absorption spectroscopy and grazing-incidence x-ray diffraction. The resultant phase diagram for the ion paired lipids sheds light on the mixing behavior of lipids in monolayer models of resistant phenotype bacterial membranes, and provides a platform for future biophysical studies. The aminoacyl lipids produced by a wide range of bacteria are becoming increasingly recognized as clinically relevant virulence factors, due to the role they play in phenotypic adaptations to the physical and biochemical stressors which confer intrinsic defence against infection.The most widely studied of these lipids is lysyl-phosphatidylglycerol (LPG), for which the genomic regulation and biosynthetic pathways in Staphylococcus aureus, have recently been elucidated in some detail.Data from microbiological assays, has shown that an increased proportion of LPG in S. aureus membranes correlates with resistance to both host defensive peptidesand membrane-active therapeutic antibiotics.The mechanisms facilitating such resistances are assumed to involve the tuning of target membrane physical properties, notably those of interfacial charge and lipid ordering, which are influenced by the LPG content in the bacteria membranes.Biophysical investigations into these phenomena have been hampered by the labile nature of native LPG, which is readily hydrolysed under mild conditions, therefore exposing any such study to the risk of artefact.To this end, stable LPG analogues have been synthesized. One such analogue, lysyl-phosphatidylethanolamine (LPE) exhibited an inhibitory effect on antimicrobial peptide activity when incorporated into vesicles containing phosphatidylglycerol (PG).A second analogue, 3-aza-dehydroxy lysyl-phosphatidylglycerol (3adLPG), facilitated enhanced membrane ordering and antimicrobial peptide resistance when mixed with PG under mildly acidic conditions,which are known to promote PG/LPG ion pair formation in model bacterial membranes.In order to gain a better understanding of the influence of lipid ion pairing on membrane structure and interfacial properties, we conducted a high-resolution study of biomimetic mixtures of dipalmitoyl-3adLPG and DPPG in Langmuir monolayers. The natural phospholipid composition [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref] observed in different S. aureus strains is dominated by PG ( � 40-70 %) and LPG ( � 30-55 %) with saturated and iso-branched fatty acids. Cardiolipin (CL) is present to 4-9 %.To simplify the lipid composition for the purposes of our biophysical investigations, only binary mixtures of DPPG and DP3adLPG [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref] were studied. Since CL is fully deprotonated at physiological pHand is known to promote negative curvature,its exclusion from the monolayer model removed the possibility that it might cause distortions in the chain packing. The use of lipids possessing only palmitoyl chains allowed us to focus on the charge interaction between the head groups, using total reflection x-ray fluorescence (TXRF), and to determine a phase diagram for condensed monolayers using grazing incidence xray diffraction (GIXD). In all of our experiments, premixed lipid solutions were spread as Langmuir monolayers at the gas/liquid interface on a pH 7.4 solution with the addition of 1 mM CsBr for the TRXF experiments. In earlier work,we encountered the problem that the ultrapure water used in the subphase contained traces of calcium. To avoid the competition of divalent calcium ions with the monovalent cesium ions for interaction with the negatively charged lipid phosphate groups,50 μM EDTA was added. At pH 7.4, DPPG is assumed to be fully ionizedwhereas DP3adLPG can be either zwitterionic or positively charged, in the same way as the native LPG [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref].To make comparisons between the charge states of the different DPPG/DP3adLPG mixed monolayers, TRXF measurementswere performed at a surface pressure of 30 mN · m À 1 [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] , the physiological lateral pressure in membranes,when all of the different compositions were in the condensed phase [fig_ref] Figure 3: A [/fig_ref]. [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] and B show the L α and L β bands of the subphase Cs + and the K α signal of Br À , both of which exhibited high intensities, allowing quantitative evaluation of the integrated values [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref]. The pure DPPG monolayer has a high negative charge which attracts of Cs + ions to the interface. The addition of DP3adLPG at a 0.33 mole fraction reduces the charge and therefore the amount of attracted Cs + . At x DP3adLPG = 0.5 a charge neutral monolayer would expected if both amino groups of DP3adLPG were protonated and full ion pairing occurs.However, the residual negative charge of the monolayer proves that this is not the case. Assuming that DPPG is fully deprotonated at pH 7.4 [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref] ,at x DP3adLPG = 0.5 half of the total lipid molecules would be expected to carry a net negative charge. The residual negative charge in the system could only result from the presence of a mixture of positively charged DP3adLPG (phosphate deprotonated and both amines protonated) and the zwitterionic species (phosphate deprotonated, ɛ amine protonated and α amine uncharged) [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref]. The small irregular shapes of the condensed phase domains observed in the 1 : 1 DPPG/DP3adLPG monolayer (Supporting Information [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] are also indicative of a charge imbalance in the lipid mixture. The extrapolated point of zero charge is at x DP3adLPG = 0.561 (Supporting Information [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref]. Examining the evolution of the bromide signal as a function of x DP3adLPG supports the same interpretation [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] , where monolayers with an x DP3adLPG > 0.5 attract Br À to the interface in proportion with DP3adLPG concentration. At x DP3adLPG = 0.5 and lower, the signal is negative because of the repulsive forces between the negatively charged monolayers and bromide which reduces the bromide concentration at the interface below the buffer value. Extrapolating the decrease in Br À intensity (Supporting Information [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref] suggests that the interface would be neutralised at x DP3adLPG = 0.524, a value slightly lower than that estimated from the Cs + signal. Averaging both values implies that approximately 15 % of the DP3adLPG was zwitterionic, with the remaining 85 % carrying a net positive charge. Using this ratio of the different protonation states, the Henderson-Hasselbalch equation gives a predicted DP3adLPG α-amine pK a of 8.15. Due to the sensitivity of the TXRF measurements, it can be readily seen [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] that when one charged lipid is in the minority, its corresponding counter ion is repelled from the interface. This clearly indicates the formation of a neutral ion paired compoundbetween the PG and 3adLPG, when the latter carries a net positive charge [fig_ref] Figure 1: The natural lipid composition of the bacterial cell membrane of S [/fig_ref]. In this respect, 3adLPG is both structurally and functionally analogous to the native bacterial lipid.aureus. The major components are phosphatidylglycerol (PG), lysyl-phosphatidylglycerol (LPG), and cardiolipin (CL).The numbers represent the pK a values according to literature for LPG.The components of the model system for biophysical investigations: 1,2-dipalmitoyl-sn-phosphatidylglycerol (DPPG) and 1,2-dipalmitoyl-sn-3-aza-dehydroxy lysyl-phosphatidylglycerol (DP3adLPG). C) Predicted charge states of LPG at pH 7.4 according to the pK a value of the α-amine given in A (calculated using the Henderson-Hasselbalch equation). The extent to which ion pairing between DPPG and DP3adLPG influenced their packing behaviour was probed using a number of monolayer techniques in addition to GIXD. Langmuir isotherms show that both the pure lipids and their various mixtures exhibit a first-order phase transition from the liquid expanded (LE) to a liquid condensed (LC) phase [fig_ref] Figure 3: A [/fig_ref]. The transition is characterized by a pronounced LE/LC coexistence region (see fluorescence microscopy images in Supporting Information [fig_ref] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region [/fig_ref] and infrared reflectionabsorption spectroscopy experiments in [fig_ref] Figure 3: A [/fig_ref]. The transition pressures of the mixtures are clearly lower compared with those of the pure lipids , with the DPPG/ DP3adLPG 2 : 1 mixture exhibiting the lowest value. This suggests that the 2 : 1 mixture forms condensed phases more readily than the other mixtures, a phenomenon which has hitherto not been observed for native LPG, as a previous study on PG/LPG mixed monolayers used lipids which did not undergo first-order transitions.The in-plane structure of the condensed phases was determined at the Angstrom level by GIXD. The monolayer of DPPG is characterized by three diffraction peaks above the horizon (Q z > 0) in the wide-angle region (at high Q xy ) at all the lateral pressures investigated. Three Bragg peaks are typical for an oblique lattice structure with tilted chains [fig_ref] Figure 3: A [/fig_ref]. The Bragg peak positions, their full-widths at half-maximum (FWHM) and all lattice parameters obtained for DPPG, DP3adLPG and the different mixtures at 20°C and different surface pressures are listed in Supporting Information Tables S1-S5. The crosssectional chain area of A 0 = 19.7 Å 2 indicates reduced rotational freedom, but it is slightly larger compared with the reported values of DPPG on water containing 1 mM CsBr.Plotting 1/ cos(t) vs. the lateral pressure and extrapolating to zero tilt angle allows the determination of the tilting transition pressure.The extrapolated value (69.6 mN · m À 1 ) is too high to be experimentally determined. Interestingly, this value is much larger compared to the one determined on a water subphase (50.5 mN · m À 1 ).This shows clearly that on water (pH~5.8) with 1 mM CsBr, DPPG is only partially ionized. However, the ionization degree depends not only on the subphase pH but also on the charge state of a mixing partner. In mixtures with DHDAB (positively charged molecule), the ionization degree of DPPG is interestingly higher compared with the pure monolayer.For this reason, we chose to conduct our experiments at pH 7.4, in order to avoid the presence of protonated DPPG. DP3adLPG exhibits a higher LE/LC transition pressure compared to DPPG [fig_ref] Figure 3: A [/fig_ref]. The head group is quite large leading to a larger molecular in-plane area of 48.0 Å 2 (at 30 mN · m À 1 ) compared to 45.2 Å 2 for DPPG. The lattice structure is the same (three diffraction peaks of an oblique lattice structure) but the cross-sectional area of the chains is with 20.0 Å 2 larger and typical for a rotator phase, as a result of accommodating the bulky head group. Two of the DPPG/DP3adLPG mixtures, 1 : 1 and 1 : 2, exhibit the same oblique in-plane lattice structure. However, the mixture DPPG/DP3adLPG 2 : 1 displays only two diffraction peaks characteristic of a rectangular in-plane lattice. The chains are tilted in the direction of the nearest neighbour (NN). Based on isotherm and GIXD data, a putative phase diagram has been constructed . The exact dimensions of the two-phase co-existence regions are unknown and would require many more mixtures to be studied what was clearly not the aim of this work. But it indicates the formation of a congruent melting compound in the binary system. The two lipids DPPG and DP3adLPG are completely miscible in the liquid-like LE phase. However, the miscibility is not ideal as the molecular area vs. mole fraction demonstrates. The molecular area in the mixtures is clearly much smaller compared to the expected one of ideal mixtures or completely de-mixed systems indicating preferred interactions between the two unlike compounds. This suggests that even in the fluid state, the lipids are associated through head group-driven ion pairing, which may explain the ordering effect these associations confer on fluid phase bilayers.In the condensed phase of the monolayer, the congruent melting compound DPPG/ DP3adLPG 2 : 1 forms an L 2 phase separated from the oblique phases on both sides of the phase diagram by small miscibility gaps. This indicates that in addition to the proportion of DP3adLPG regulating the surface charge through ion pairing with DPPG, it also alters chain packing and monolayer condensation, but in a somewhat unexpected way. Although the near surface neutrality of the 1 : 1 mixture demonstrates a higher degree of ion pairing and thus presumably a more condensed and stressor resistant combination,it is the 2 : 1 mixture which, although more anionic, appears to have the greater propensity to form a stable condensed phase. How this proportion of the lipids might affect bacterial physiology in nature requires further study, however, it should be noted that for a number of S. aureus strains, when grown at physiological pH, the proportion of anionic to cationic lipids in their membranes is approximately 2 : 1.Despite the differences in lattice geometries between the 2 : 1 and the other DPPG/DP3adLPG mixtures, in all cases the tilt angle of the chains decreases with increasing lateral pressure . Comparing the tilt angles at 30 mN · m À 1 displays an interesting behaviour of the mixtures . The tilt angle is quite constant over a wide composition range showing that the thickness of the membranes does not depend on the composition. Therefore, despite alteration in membrane charge and condensation potential, altering DP3adLPG content should not affect the thickness of bilayers in which it is combined with DPPG. It should be noted that due to the differences in the calculated α-amine pK a of native LPG (pK a 7) and DP3adLPG (pK a 8.15) the pH conditions modelled in this study would be comparable to the native lipid at pH 6.25 (according to the Henderson-Hasselbalch equation). In S. aureus, the proton gradient across the plasma membrane ensures that the outer leaflet has a pH of 6.25-6.65,which means that our monolayer model provides an accurate mimetic for the environment-facing half of the lipid bilayer. Thus, 3adLPG is not only useful in biomimetic model membranes used to assess antimicrobial peptide mechanisms against S. aureus,but should also fulfil the need for more biorelevant lipid environments for the biophysical study of bacterial membrane proteins.It is increasingly recognised that in order to improve our knowledge of the preventative and therapeutic interventions which can be made in host/pathogen interactions, a multidisciplinary approach is required. Bacterial membrane biophysics can play a very important role in this field, by contributing to research elucidating the mechanisms of susceptibility and resistance to novel therapeutics such as antimicrobial peptides,and facilitating structure and functional studies on membrane-associated bacterial virulence factors. The validity of such biophysical research relies on the biological relevance of the model systems employed, which becomes more important as they necessarily increase in complexity, to improve their biomimetic proximity. One way to ensure this would be to use lipids extracted from bacteria.However, for studies which require membranes of defined composition whose physical properties may be tuned to suit specific purposes, especially those related to S. aureus and other aminoacyl lipid containing pathogens, 3adLPG may prove to be a valuable tool. ## Experimental section Experimental details are given in the Supporting Information. . A) Phase diagram (lateral pressure π versus the mole-fraction of DP3adLPG) of the DPPG/DP3adLPG system. The phases observed by GIXD are obl (oblique), L 2 (orthorhombic with NN tilt). The first-order transition pressures (*) from the disordered LE to an ordered LC phase are determined by pressure-area isotherms. B) Tilt angle t at 30 mN · m À 1 (the lateral pressure in biological membranesversus the mole fraction x DP3adLPG . [fig] Figure 1: The natural lipid composition of the bacterial cell membrane of S. [/fig] [fig] Figure 2: Sections of the buffer corrected TRXF spectra of the caesium region (A) and the bromide region (B) of Langmuir monolayers composed of DP3adLPG, DPPG and different mixtures on a subphase adjusted to pH 7.4 containing 1 mM CsBr and 0.05 mM EDTA. C) Intensities of the L α and L β bands of the Cs signal (grey shaded area in A: L α = 4.286 keV, L β1 = 4.619 keV, and L β2 = 4.935 keV) and the K α signal of Br (grey shaded area in B: K α = 11.924 keV) as a function of the mole fraction of DP3adLPG. [/fig] [fig] Figure 3: A) Pressure-area isotherms of DPPG (red), DP3adLPG (black) and the DPPG/DP3adLPG mixtures 2 : 1 (blue), 1 : 1 (green) and 1 : 2 (magenta) on a pH 7.4 subphase containing 1 mM CsBr at 20°C. B) GIXD data (scattered intensity vs. the vertical and horizontal components of the scattering vector) of the DPPG/DP3adLPG 2 : 1 and 1 : 1 mixtures at 30 mN · m À 1 and the fitted Bragg peak and rod positions (black crosses). The width and the length of the crosses represent the FWHM of the peak and the corresponding rod. [/fig]

Memory and communication efficient algorithm for decentralized counting of nodes in networks Node counting on a graph is subject to some fundamental theoretical limitations, yet a solution to such problems is necessary in many applications of graph theory to real-world systems, such as collective robotics and distributed sensor networks. Thus several stochastic and naïve deterministic algorithms for distributed graph size estimation or calculation have been provided. Here we present a deterministic and distributed algorithm that allows every node of a connected graph to determine the graph size in finite time, if an upper bound on the graph size is provided. The algorithm consists in the iterative aggregation of information in local hubs which then broadcast it throughout the whole graph. The proposed node-counting algorithm is on average more efficient in terms of node memory and communication cost than its previous deterministic counterpart for node counting, and appears comparable or more efficient in terms of average-case time complexity. As well as node counting, the algorithm is more broadly applicable to problems such as summation over graphs, quorum sensing, and spontaneous hierarchy creation. OPEN ACCESS Citation: Saha A, Marshall JAR, Reina A (2021) Memory and communication efficient algorithm for decentralized counting of nodes in networks. PLoS ONE 16(11): e0259736. https://doi.org/10. # Introduction All decentralized systems share the common aspect of being comprised of a network of units (which can be considered as graph nodes) that rely on local and partial information which they can gather from the subset of devices in their communication range (communication links can be represented as graph edges). An open challenge is to allow the units of these largescale decentralized systems to estimate properties of the entire group. A fundamental property that is crucial for the design and the efficient functioning of several systems is the system size, that is, the number of units in the system. Computing the exact network size in finite time with a decentralized algorithm with finite complexity is proved to be impossible. Previously proposed solutions are therefore stochastic algorithms that only give an approximation of the system size, providing the possible advantages of robustness and speed. Deterministic algorithms provide the exact solution in a finite time, however, they may rely on stringent assumptions on the communication network topology. An overview of the existing algorithms is provided in Section State of the art. We propose, in Section The aggregate-and-broadcast algorithm, a new decentralized deterministic algorithm, the aggregate-andbroadcast (AnB) algorithm, that iteratively aggregates the node counts into a small number of local hubs which finally broadcast the count throughout the whole network. The AnB algorithm allows the nodes to compute the exact network size in a finite time when an upper bound is provided. In other words, the network size computed by the AnB algorithm is exact up to a limit that is bounded by the algorithm's execution time, as proved in the S1 File. The algorithm relies on the only two assumptions of a connected network and uniquely identifiable units (i.e. unique id), and requires minimal computation and communication capabilities of the units. The algorithm performance is analyzed and when possible compared with previous algorithms in terms of time, communication, and memory costs (see Section Analysis of the algorithm). The results indicate that the AnB algorithm is scalable, efficient, and accurate, with better performance than the existing algorithms in terms of smaller memory and communication costs. Therefore, as discussed in the Conclusion, the AnB algorithm can be beneficial for systems with constrained memory and communication, and has the potential to be employed in numerous application cases and impact a large variety of decentralized systems. # The problem statement Consider a connected network G ¼ ðV; EÞ, where V ¼ f1; . . . ; Ng is the set of nodes in the network and E � V � V is the set of the edges of the network. The edges describe undirected and unweighted communication links between nodes, i.e. , (v, u) 2 E. Each node can only communicate at synchronous timesteps with its neighbors, where the set of neighbors of the generic node v is defined as N i ¼ fu 2 Vjðv; uÞ 2 Eg. We assume G to be time-invariant. Each node is characterized by a unique identifier (id). Each node knows an upper bound N max of the network size, such that N max � N. In this paper, we propose an algorithm to be executed by every node of the network to allow them to compute the network size N in a finite number of iterations t max � 4N max + 1 (and therefore, a finite amount of time). Note that knowledge about N max is only necessary in order to bound the number of iteration steps required for the execution of the algorithm to t max . This is required due to the results reported by Hendrickx et al.who have proved that it would be otherwise impossible for a finite complexity algorithm to correctly count the number of nodes (see discussion in Sec. Stopping criteria). ## State of the art Most of the algorithms proposed to estimate the size of the network rely on stochastic methods. The most common approach relies on executing variations of random walks on the network. In particular, Ganesh et al.used continuous time random walks to obtain a target number of redundant node samples. The time required to obtain such a sample was then used to estimate the network size. In a different study, Gjoka et al.compared various weighted random walk techniques. The study identified efficient methods to identify various macroscopic properties of the network by simulating weighted random walks on the network (e.g. Metropolis-Hastings Random Walk and Re-Weighted Random Walk). Similarly, Katzir et al.proposed a method based on simulating multiple simultaneous random walks in order to estimate the size of the network. Building upon this work, Musco et al.proposed an algorithm where multiple nodes execute random walks and compute the network size based on the degrees of the nodes encountered. Notable stochastic algorithms which do not involve random walks rely on either average consensusor on order statistics consensus. One of the shortcomings of stochastic algorithms is that their run-times depend on the desired accuracy of the results. Therefore, for applications where the size of the network is required to a high degree of accuracy, stochastic algorithms might take a long time to converge. For instance, the number of dynamical attractors in Boolean networks and their periodicities depend on whether the network size is even or odd, prime or composite. Since dynamics on such networks are crucial in studying social networks, neural networks and gene and protein interaction networks, accurate knowledge of the network size is crucial. In such scenarios, deterministic algorithms to estimate the network size are better suited. To the best of our knowledge, the number of deterministic algorithms for decentralized network node counting is very limited. One of the most trivial algorithms is the All-2-All method, as alluded to in Ref.. It consists in having each node broadcasting a unique id together with all ids that it has already received so far. This simple algorithm is the most efficient algorithm we are aware of for deterministic network node counting on general network topologies. Other algorithms for node counting have been proposed for networks with specific topologies. For example, an algorithm inspired by the Breadth-First-Search (BFS) algorithm can be used on a tree network. In 2003, Bawa et al.generalized such an algorithm so that it could be implemented on a network with a general topology. In their paper, the authors propose three different algorithms which may be used for computing various aggregates across the network. While the proposed algorithms are efficient, they investigated a different problem. They focus on the situations when the network size or the other aggregate quantities are sought by a single node of the network. When every node requires the size information, repeating the algorithm ofon every node becomes less efficient than the All-2-All method, as described in Sec. Analysis of the algorithm. Notably, numerous algorithms have been proposed to create a spanning tree on a general network. However, they are constrained in a crucial aspect as underlined in the next section. ## Significance of the work In Ref., the authors propose algorithms to create a spanning tree on the given network. Once a tree is constructed, any information can be aggregated in the root by following the edges of the tree. Due to its important applications, numerous other algorithmshave also been proposed to construct a spanning tree on a connected network. All these algorithms, in order to build a spanning tree, require that one node of the network assumes the role of the root of the tree. However, selecting one node to assume such a role through a decentralized algorithm running on a sparse network is a difficult problem on its own. In fact, the network nodes would need to invest resources (time and computation) to reach a consensus on a single root node and avoid duplicates. In this paper, we present an algorithm to create a 'tree-like' network to span a general connected network without assuming any particular node as a root. Instead of generating a tree from the root, our algorithm removes edges consecutively based on the local neighborhood of each node. This results in the emergence of possibly multiple 'root-like' nodes (which we call 'residue' nodes). Any information which was initially distributed among all nodes of the network can therefore be concentrated in these residue nodes. Thereafter, the information can be broadcast throughout the network. In addition to relaxing the restriction of a selected root, the AnB algorithm performs better than the other known algorithms in terms of communication and memory costs than the existing algorithms. In fact, typically, the AnB algorithm, by creating multiple root-like nodes, decentralizes the computation to different parts of the network and thus nodes use on average less memory and send fewer messages. Our empirical analysis shows that the time costs of the algorithm depend crucially on the network topology: the proposed algorithm performs better than the previous algorithms for large random geometric networks but worse than them for other types of network topology. Hence, the AnB algorithm may prove to be useful in networks where the required memory per node is the major limiting factor or the limited communication between nodes is desirable. Additionally, the proposed algorithm can be used to perform other collective tasks where aggregation of information is required but a distinguished root node cannot be identified (see the Conclusion). ## The aggregate-and-broadcast algorithm We propose the aggregate-and-broadcast (AnB) algorithm, a deterministic algorithm for the simultaneous and decentralized determination of the size N of a finite connected network by all its nodes. We assume that each node of the network has a unique id, can communicate only with its immediate neighbors, and knows N max , the upper bound of the network size. Other than that, we make no prior assumptions about the topology of the network nor prior knowledge of the node. The underlying idea of the AnB algorithm is inspired by the standard nodecounting method on a tree by its root. In a tree, the counts of the leaves are assimilated by their respective parents and then the leaves are iteratively pruned. Applying such an algorithm on a graph with a general topology poses a challenge since a strict hierarchy does not exist among the nodes. To overcome this problem, we add a step in each iteration where, based on the degree of its neighbors, each node determines its local hierarchy which, in turn, determines whether it should be pruned or not. In the next subsections, we describe the proposed AnB algorithm in detail. We start with an overview of the entire algorithm in the next subsection. In subsections Pre-iteration steps and Iteration steps, we describe the pre-iteration steps (which include variable initialization) and the iteration steps of the algorithm respectively. Finally, in subsection Remarks on the AnB algorithm we compare the AnB algorithm to the standard node counting algorithm in trees and make some further remarks about the proposed algorithm. The correctness of the AnB algorithm is proved in Sec. Theorems and Proofs of the S1 File. ## An overview of the anb algorithm Prior to the iterative steps, the nodes of the network are initialized as follows. The behavior of a node with id i at any particular instant is determined by its state s i which can take one of four values during the course of the algorithm: 'active' (A), 'leaf' (L), 'residue' (R), or 'inactive' (I). The state of each node is initialized to s i = A. Each node also starts with a local node counter c i = 1. Since, at the beginning of the algorithm, each node is aware only of its own existence, the counter is initialized to 1. As the algorithm progresses, the node gathers information about the changing state of nodes (equivalent to the nodes getting 'pruned') from its neighbors and updates the value in c i . Additionally, each node also has the following other internal variables: the set of its neighbors N i , its effective neighborhood E i , effective degree e i , the set of residues R i and final node count n i . Among these, the first three variables are initialized to be empty [formula] sets N i ¼ E i ¼ R i ¼ ; [/formula] , and the effective degree and final count variable are initialized as e i = n i = 0. Note, that it is assumed that the nodes of the network are synchronised and have a common sense of time. In other words, the nodes are aware of the beginning and end of each iteration step of the algorithm. Therefore, implementation of the AnB algorithm on a distributed system, e.g., a robot swarm or sensor network, will also need a mechanism to guarantee that synchronization is achieved and maintained. From the perspective of a node, the AnB algorithm is divided into two phases: 'pre-reduction' and 'post-reduction'. A node is said to be in pre-reduction phase when its state is either s i = A or s i = L. As this phase progresses, a node in 'active' state updates its local counter c i by locally accumulating information from 'leaf' neighbors getting 'pruned' until the node itself changes its state to s i = L and becomes a 'leaf' node. Note that, here the term 'leaf' is used to denote a node which is about to be 'pruned' from the network; and not necessarily a node with only one neighbor. In the next iteration, each leaf node, depending on their effective neighborhood E i , again changes its state to either (a) s i = I and gets 'pruned', or (b) s i = R and becomes a residue node. At the end of pre-reduction phase, the nodes of the network are either in residue (s i = R) or inactive (s i = I) states. These states can be considered analogous to the 'root' and the 'pruned leaves' of a tree network respectively. The residue nodes contain parts of the total count of nodes in the network. This is similar to the root of a tree network which contains the total node count of the entire tree after all the nodes have been pruned. This information is then broadcast across all other nodes and assimilated to give the final node count of the network. To do this, each residue node constructs a 'broadcast message' b i , sends it to all its neighbors and changes its state to s i = I. This broadcast message is then relayed by all nodes-irrespective of their state s i -across the network. A node that receives a broadcast message adds the partial count to its final count variable n i , and keeps track of the residue nodes to avoid double counting. Thus, after iteration steps t max , the variable n i gives the total count of all nodes in the network. Further details of the algorithm and the the stopping criteria are provided in subsections Iteration steps and Stopping criteria respectively. ## Pre-iteration steps We now describe the AnB algorithm in detail. The actions taken by a node i in a particular step are determined by its internal variables and the messages it receives from its neighbors, i.e. the nodes in N i . Any message sent by a node is denoted as m i,h , where i is the sender of the message and h is the 'type' of the message. The 'type' of the message determines the action to be taken by the receiver of the message. The various types of messages and their roles are summarized in. Note that every message is broadcast to the entire neighborhood N i and thus, can be accessed by all nodes in N i . After the initialization of all internal variables, each node of the network identifies its neighborhood. To do so, it sends a message m i,echo indicating its presence to all its neighbors. It then receives similar messages m j,echo from other nodes. The set of all nodes from which such a message is received is then identified as the neighborhood N i (Line 4). One of the most crucial internal variables for the node is its effective degree e i which is the number of its neighbors which are in the active state (s i = A). Since all nodes start in the active state, the initial effective degree of the node is the number of elements in its neighborhood: e i ¼ jN i j. In addition to its own effective degree, the node also needs to be aware of the effective degrees of those neighbors which are in active state. The node keeps track of this information in form of its effective neighborhood, [formula] E i ¼ fðj; e j Þ : j 2 N i and s j ¼ Ag:ð1Þ [/formula] Therefore, E i is a set of tuples where the first element of the tuple is the id of an active neighbor of i and the second element is the effective degree of the neighbor. The identification of neighborhood also allows the node to compute its initial effective degree e i ¼ jN i j and to send it to its neighbors as m i,degree . Thereafter, a node i receiving a message m j,degree updates its effective neighborhood E i as described in Line 8. ## Iteration steps After the pre-iteration steps, the node i enters an iterative phase where its steps are determined by its state s i . The details of these state-dependent steps are illustrated in the finite state machine ofare elaborated as follows. - Active nodes: Each active node i with s i = A first detects any change in its neighborhood. This change can be of two types: (a) Either some of its neighbors are transitioning to inactive state (message with h = count); or (b) the effective degree of some of its neighbors is being reduced (message with h = reduce). Therefore, upon receipt of a message m j,count , the node i excludes the sender from its effective neighborhood E i , decreases its effective degree e i by 1 and assimilates the contents of the message in its local count (Line 14), Since the effective degree of node i is decreased by 1, it sends a message m i,reduce to its neighbors. For each message of type h = 5 received, the node updates the record of the effective degree corresponding to the sender of the message (Line 19). After processing the incoming messages, the node i checks for the two conditions indicated in Line 20. If both conditions are met, the node sends a message m i,leaf and changes its state to s i = L; otherwise, the node stays in the active state for the next iteration. [formula] c i ¼ c i þ m j;count :ð2Þ [/formula] - Leaf nodes: The node i in state s i = L stays in this state for exactly one iteration and then changes its state to either s i = R or s i = I. First, it processes any incoming message of the type h = leaf. The reception of any such message implies that some of its neighbors have transitioned to the leaf state in the same time step, and are therefore no longer in the active state. For each message m j,leaf received, the effective degree e i of the node is reduced by one. After processing all incoming messages, the node i changes its state; if the effective degree e i = 0, it change its state to s i = R otherwise, it sends the message [formula] m i;count ¼ c i e ið3Þ [/formula] and changes state to s i = I (Lines 27-31). - Residue nodes: Each node i in state s i = R updates its residue set R i with its own id i and the total node counter n i adding its local counter c i . It then broadcasts a message m i,broadcast = (i, c i ) and changes its state to s i = I. - All nodes: While the previous steps are executed by nodes in a specific state, the following steps are executed by all nodes of the network at each iteration irrespective of their state. Whenever a node i receives a message m j,broadcast = (k, c k ) from any of its neighbors, it checks if node k is in the residue set R i . If k= 2R i , the node i adds k to its residue set [formula] R i ¼ R i [ fkg, [/formula] adds the corresponding local count c k to its final node count n i = n i + c k and finally relays the message forward by sending message m i,broadcast = m j,broadcast . After a sufficient number of iteration steps t max , all nodes converge to the same final count n i equal to the network size N. A detailed analysis of the convergence time is provided in Sec. Time Cost of the S1 File. An illustration of the working of the aggregate phase of the AnB algorithm is shown in ## Stopping criteria The AnB algorithm terminates when sufficient iteration steps, t max , has passed. This t max should be sufficiently large so that each broadcast message reaches every node of the network. However, determining an exact value for t max is impossible as reported by Hendrickx et al.who have shown that it is impossible for a finite complexity algorithm to correctly estimate the size of a network with probability one. If t max could be exactly determined for the network, we would be absolutely sure that each residue message has reached every node and hence, each node is aware of the size of the network. This would be in direct violation of the aforementioned result. However, depending on the prior knowledge about the network, various estimates of t max can be made as follows. In Sec. Theorems and Proofs (Corollary 1) of the S1 File, we show that the maximum time required for all nodes to reach the final state, i.e., the inactive state, has the above boundary of t r = 3N + 2. It is also trivial that the number of time steps required to broadcast a message across a network of size N is, in the worst-case, t b = N − 1. Therefore, t max is bounded above by t r + t b = 4N + 1. Hence, if an overestimate N max of the network size is known apriori, we can set t max = 4N max + 1 to know the exact size of the network in finite time. ## Remarks on the anb algorithm As shown ina node spends exactly one iterative step as a leaf, and at most one iterative step as a residue node. Therefore, a typical node spends most of its iterative steps in either active or inactive states. We can now elaborate on the similarities and differences between the proposed AnB algorithm and the standard node-counting method on a tree network which were indicated earlier. On comparion, we note the following points of interest. 2. In a tree network, leaves are easily identified as nodes with degree one. Since this is not true for a general network, we use the condition in Line 1 to identify, at each iteration step, the nodes which are to be labeled as leaves. 3. After a node has been identified as a leaf in a tree network, it passes on its local count to its parent and gets transformed to a pruned leaf. In a tree network, the parent of each node is unique. However, in a general network, a leaf node may have more than one parent. Therefore, in the AnB algorithm, the local count of each leaf is divided equally among all parents to avoid over-counting number of nodes. 4. Once the counts have been passed on, the leaf node becomes an inactive node, similar to the pruned leaves in a tree network. If there are no active neighbors ('parents') to which a node can pass on its local count, it becomes a residue node, which is similar to the root of the tree. While the structure of the tree implies that there can be only one root of a tree, there is no such restriction for a general network. Hence, the count of the size of a general network gets concentrated into the residue nodes which is then broadcast and recombined in the final stages of the AnB algorithm. It is to be noted that each node checks for the reception of a message of type h = broadcast at each iteration. This is necessary because messages of type h = broadcast carry the node count of a part of the network as counted by a residue node. Therefore, all nodes which receive such a message should add it to their final count and send it further. This is in contrast with the other types of messages which are intended only for nodes in active or (as in case of h = leaf) leaf states. ## Analysis of the algorithm In this section, we demonstrate the correctness of the AnB algorithm and analyze the algorithm performance in terms of time, communication, and memory costs against the known node-counting algorithms. We do not compare AnB with stochastic algorithms which only compute an estimate of the network size that increases over time, but we limit our comparison against algorithms that return the exact node count in a finite time: the All-2-All algorithm and the Single Tree (ST) algorithm. The All-2-All algorithm is, to the best of our knowledge, the only known deterministic algorithm for node counting which can work on any type of connected network regardless of its topology. In the All-2-All algorithm, each node broadcasts its id, and all received ids, to all its neighbors and every node counts the number of received unique ids. The ST algorithm, instead, is the most efficient of the three algorithms proposed by Bawa et al. in. Despite being stochastic, the ST algorithm is proved to return the exact network size in a finite time. The ST algorithm, similarly to AnB, relies on the construction of a treelike hierarchy. However, in its original form, the ST algorithm allows only a single node to compute the network size. In order to allow all the nodes of the network to know the network size, the ST algorithm can be extended in the following two ways: (a) one randomly selected node executes the ST algorithm and then broadcasts the computed size to all other nodes; or (b) all the nodes of the network simultaneously execute the ST algorithm and compute the network size independently. Employing alternative (a) requires the nodes to be able to select in a decentralized way which node will execute the ST algorithm. Decentralized node-selection adds a new problem which may require further assumptions on the network topology or on the initial knowledge of the nodes. Therefore, in our comparison against the ST algorithm, we employ alternative (b) by which every node makes an independent count of the network size. We provide a comparison both as worst-case algorithm complexity and with generic analytical equations for each type of cost. When such analytical solutions are not possible, we provide the results of numerical simulations for specific graph topologies. In fact, the AnB algorithm is proved to work on any connected graph regardless on the graph topology. Through our analysis, we highlight the differences in performance for each topology. ## Correctness of the anb algorithm In Sec. Theorems and Proofs of the S1 File, a detailed proof of correctness of the algorithm is provided. A brief sketch of the proof is as follows. We begin by identifying a sequence of time steps of the algorithm when the variables e i and E i correctly give correct information about the neighborhood of the node i (see Theorem 1). We say that, at these time steps, the network is in the resting state. We then show that, as the network progresses from one resting state to another, the number of active states decreases. During this process, the information about their local node counts c i gets concentrated into the nodes which pass through the residue state (see . Therefore, when no active nodes are present in the the network, the information about the size of the network is concentrated in the nodes which passed through the residue state. This information is then broadcast throughout the network and is accumulated by each node (see Theorem 3). ## Comparison with other algorithms in terms of complexity We compare the efficiency of the AnB algorithm against the All-2-All and the Single Tree (ST,algorithms in terms of three aspects: (a) the time required to compute the network size by every node, (b) the number of messages sent by all nodes (i.e. the communication cost), and (c) the minimum amount of memory required by each node to execute the algorithm (i.e. the memory cost). Note that, it is difficult to compare the efficiency of AnB against most other stochastic algorithms because their efficiency depends on the desired accuracy of the results. The more accurate we want the results to be, the longer the stochastic algorithms should run, at the cost of increased time and/or communication costs. On the other hand, deterministic algorithms like ours give accurate results in a finite time and make possible asymptotic performance analysis. The efficiency results for the AnB algorithm are derived in Sec. Complexity Analysis of the S1 File and reported in . We derive exact results for the communication and memory costs. Instead, computing a precise equation of the time cost is difficult, as it depends strongly on the topology of the network which evolves at every time step (see discussion in Sec. Time Cost). Through Theorem 3 in Sec. Theorems and Proofs, we computed the upper bound of the time complexity of AnB. To analyse the exact performance in terms of time, instead, we computed a set of numerical simulations on various graph topologies whose results are shown inIn particular, we implemented and tested the AnB algorithm on four different types of random networks as listed in. The results of our analysis show a qualitative difference in algorithm performance as a function of the network topology. We employed these numerical simulations to compare the temporal performance of AnB with the All-2-All algorithm and to make general considerations on the execution time of the AnB algorithm (see also Sec. Time Cost). . Exact costs for the two algorithms for a general network with diameter D, average degree d, and r residue nodes. For memory cost, we indicate the individual degree d i for the generic node i. The AnB algorithm is more efficient than the All-2-All and the ST methods in terms of memory and communication. Analytical solution for time is out of reach and we provide numerical results in ## Algorithm time communication memory AnB numerically in. The diameter is known up to a scaling factor, here we report curves scaled to values comparable to AnB's execution time to ease the comparison. In fact, the intersection of same-colour curves indicates that for large networks, the AnB algorithm is asymptotically slower than the All-2-All method. This is the case for all the analyzed network topologies but the Random Geometric networks. In RG networks, All-2-All shows a steeper curve that would slow down the process for very large networks (see inset on a log-log scale). The right panel shows the fraction of residue nodes x ¼ r N in the network. Low x implies low r and hence better performance of AnB algorithm in terms of memory and communications cost (see . For each network size, we report the average results for the simulation of 1,000 independent random networks. (95% confidence intervals are reported in the left panel as shades but often are smaller than the line width). [formula] (4 + r + d) − r (2d i + r + 5)log(N) All-2-All D N 2 N log(N) ST 2D [/formula] https://doi.org/10.1371/journal.pone.0259736.g003 The time, communication, and memory costs for All-2-All algorithm are relatively easy to compute. In terms of time, the algorithm ends when the messages created by every node (containing its id) reach every other node. Therefore, the time steps required for this to happen is equal to the diameter D of the network. In terms of communication, since each node broadcasts the id of every node to its neighborhood, the number of messages sent by each node is N and hence the total number of messages sent in the whole network is N 2 . Finally, in terms of memory, each node needs to store the id of every node in the network. Therefore, the minimum memory required by each node is N log(N), by assuming that each id needs at least log(N) bits. The time and communication efficiency of the ST algorithm has been outlined by Bawa et al. in. We updated their efficiency measures in order to include the changes required to allow all nodes to compute the network size. Additionally, we derived the memory cost which was not originally indicated in. The details of the complexity analysis are reported in Sec. Complexity Analysis of the S1 File; the results are reported in . The results in show that the AnB algorithm has the lowest costs in terms of memory and computation compared with the All-2-All and ST algorithms (see also . The efficiency of the AnB algoritms is higher for networks which have the number of 'residue' nodes r much smaller than N. This is the case for most random networks as shown in. Our analysis also shows that the largest share of communication messages are typically sent by the residue nodes and the largest memory is typically required to store the ids of the residue nodes. Since the fraction of residue nodes is low for all the analyzed network classes, with the AnB algorithm the nodes send comparatively fewer messages and have lower memory requirements than with the All-2-All and ST algorithms. The only cases where the All-2-All and ST algorithms might perform better than AnB in terms of memory and communication are completely connected networks, almost completely connected networks, and networks with specific topologies (such as ring networks; see detailed discussion in Sec. Performance on Ring and Complete Networks of the S1 File). In terms of time,shows that the All-2-All method scales as the network diameter D and the AnB algorithm has comparable, or slightly worse, time performance. Finally, in terms of all three complexity aspects (time, communication, and memory), in the worst case (i.e., when d i = N − 1 and r = N), the AnB algorithm has an asymptotically complexity equal to the other algorithms (see in the S1 File). Therefore, we conclude that the AnB algorithm is advantageous for applications with constrained or high-cost communication and memory, as confirmed by the results reported in and # Conclusion In this paper, we propose the AnB algorithm, a deterministic algorithm by which all nodes of a network can become aware of its size. The AnB algorithm assumes no inherent hierarchy Conversely, if a node enters the broadcasting phase earlier, it is more likely to be 'peripheral'. While various other centrality measures exist for such classification of nodes in a network (for instance, closeness centralityand betweenness centrality, they generally require the computation and ordering of a measure by a centralized agency. In the proposed AnB algorithm, the nodes can spontaneously organize themselves into a hierarchy. 3. Computation of other aggregate quantities: Similar to other previously known algorithms of network size estimation, the AnB algorithm can also be used to compute other global properties across networks. For example, if each node i is associated with a property s i , they can compute the sum ∑s i by simply setting c i = s i and executing the AnB algorithm. Similarly, other aggregate quantities such as averages and maximums/minimums can also be computed by suitably adopting the AnB algorithm. Supporting information S1 File. (PDF) # Author contributions Conceptualization: Arindam Saha.

Surmounting the obstacles that impede effective CAR T cell trafficking to solid tumors Innovative immunotherapies based on immune checkpoint targeting antibodies and engineered T cells are transforming the way we approach cancer treatment. However, although these T cell centered strategies result in marked and durable responses in patients across many different tumor types, they provide therapeutic efficacy only in a proportion of patients. A major challenge of immuno-oncology is thereby to identify mechanisms responsible for resistance to cancer immunotherapy in order to overcome them via adapted strategies that will ultimately improve intrinsic efficacy and response rates. Here, we focus on the barriers that restrain the trafficking of chimeric antigen receptor (CAR)-expressing T cells to solid tumors. Upon infusion, CAR T cells need to home into malignant sites, navigate within complex tumor environments, form productive interactions with cancer cells, deliver their cytotoxic activities, and finally persist. We review the accumulating evidence that the microenvironment of solid tumors contains multiple obstacles that hinder CAR T cells in the dynamic steps underlying their trafficking. We focus on how these hurdles may in part account for the failure of CAR T cell clinical trials in human carcinomas. Given the engineered nature of CAR T cells and possibilities to modify the tumor environment, there are ample opportunities to augment CAR T cell ability to efficiently find and combat tumors. We present some of these strategies, which represent a dynamic field of research with high potential for clinical applicability. several metastatic cancers. However, the majority of patients with advanced cancers still do not experience sustained clinical benefit from immunotherapy, highlighting the presence of barriers that one needs to identify in order to design strategies that overcome them. Ineffective T cell migration and, in particular, penetration into the tumor mass might represent an important obstacle to T cell based immunotherapies. As a support for this notion, various clinical studies have shown that tumors enriched in T cells are more susceptible to be controlled by programmed cell death-1 (PD-1) blockade. In contrast, tumors with so-called "immune deserts" and immune excluded profiles, in which T cell are present within tumors but not in contact with malignant cells, are refractory to PD-1 blockade. ## T cell subsets, location, and motility in the context of natural responses to cancer The natural development of T cell responses against tumors is a highly regulated process that involves multiple steps occurring at different locations. A prerequisite is the expression of antigens, which are specific of the tumor and may result from gene mutations or aberrant gene expression.Dendritic cells (DCs) present at the tumor site may capture such tumor antigens, for example, in the context of tumor cell apoptosis, before migrating to draining lymph nodes and presenting the antigens to T cells.In particular, the migratory conventional type 1 DC subset is able to cross-present antigens to CD8 + T cells via MHC-I molecules.Alternatively, tumor cells may translocate to lymph nodes draining the primary tumor and deliver antigens directly at this site.Another possible scenario for T cell priming against tumor antigens is represented by the assembly of tertiary lymphoid organs directly at the site of tumor development.Owing to the expression of TCRs with enough affinity for the presented antigens, T cells will get activated,leading to their clonal expansion and to the maturation into effector T cells. In particular, CD8 + T cells will acquire cytotoxic function via the expression of effector molecules such as perforin and granzymes. Effector T cells will also acquire the expression of adhesion molecules and chemokine receptors allowing them to migrate to peripheral tissues.In fact, these effector cells are preferentially attracted to sites of inflammation, such as those associated with tumor growth. However, as compared to infectious settings, T cell trafficking to solid cancers might be inefficient, in part because of a mismatch in homing molecules and receptors, down-regulation of adhesion molecules, and aberrant vasculature.A side effect to this might be the relocation of tumor-specific T cells to other sites, as effector T cells can reach different inflamed tissues, regardless of the site of initial antigenic stimulation. The ability for tumor-specific effector T cells to gain access to sites of tumor metastasis appears to be crucial to the control of cancer spreadingand possibly to maintain new seeds of cancer cells in a state of dormancy.It is however unclear whether effector T cells may act inside the vasculature to control circulating cancer cells or at the site of distant seeding. The ability of immune checkpoint therapy to control the metastatic process has been documented in several clinical trials.However, whether this applies to CAR T cell therapy remains to be determined. In the case of an efficient effector T cell response, the antigenic trigger is cleared and a fraction of effector T cells develop into peripheral memory T cells that can reside in the target tissue for prolonged periods, as a means to provide protection against a secondary occurrence of the antigenic trigger. However, in the context of cancer, the tumor cells as well as their microenvironment offer resistance to the effector function of T cells and resolution is rarely achieved. This is associated with the development of T cells within tumors that exhibit exhaustion features.Exhaustion corresponds to various T cell differentiation states usually associated with chronic antigenic stimulation and with reduced function as compared to effector cells. Whether exhausted T cells may also harbor altered motility and homing properties remains to be investigated. In this context, the differentiation status of therapeutic T cells generated by in vitro stimulation and specific culture conditions is expected to highly condition their fate in vivo, including their effector functions toward the tumor and most probably their trafficking properties. Defining and generating the population of T cells with the highest potential to control tumor growth is of pivotal importance for the design of T cell based therapies. The rare subset of T memory stem cells (T SCM ) is of particular relevance, because of its self-renewal capacity and ability to differentiate into effector cells.T SCM harbor a unique combination of naïve T cell markers and memory markers such as CD58, CD95, IL-2R , and CXCR3. A cell tracking study, performed in patients having received genetically modified T cells in the context of gene therapy trials for ADA-SCID, has highlighted that T SCM can persist for up to 12 yr post-infusion and that such cells maintain they precursor potential.Single cell analysis of T cell populations infiltrating different human cancers has recently uncovered the presence of T cells endowed with some level of stemness. In the context of nonsmall cell lung cancer, stem-like CD8 + T cells, harboring a partial exhausted phenotype together with effector potential and self-renewal capacity, have been identified.However, these cells lack the canonical T SCM phenotype of circulating T cells. In favor of the notion that these cells play a protective activity, they are lost with disease progression. Analysis of a cohort of patients with kidney cancer showed that T cell infiltrates are composed of T cell factor-1 (TCF1) + stem-like and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) + terminally differentiated T cell subsets.These subsets harbor proliferative potential and cell killing potential, respectively. Furthermore, TCF1 + stem-like T cells give rise to TIM3 + terminally differentiated T cell upon stimulation. Interestingly, TCF1 + CD8 T cells are located in regions enriched with MHC-II + cells across various human cancers, suggesting that stem-like CD8 + T cells require a lymphoid-like environment and are key to sustain the terminally differentiated T cell population mediating the antitumor immune response. Another T cell subset of interest for the prospects of T cell therapy is represented by CD8 + tissue-resident memory T (T RM ) cells, which have been shown to constitute a prevalent effector population in the microenvironment of primary triple-negative breast cancer.Indeed, a T RM gene expression signature is associated with improved relapse-free and overall survival after standard chemotherapy, implying a beneficial antitumor function for this subset. In this context, such cells display reduced expression of tissue egress genes such as S1PR1 and KLF2. Residency within the tumor might therefore be considered a desired property for the design of T cell based therapies. On the other hand, CD19 CAR T cells with CD28 domain (marketed as Yescarta) have been approved for adult relapsed or refractory large B cell lymphoma. In the past few years, clinical trials using these CAR T cells on malignant/leukemic B cells have shown high rates of response (70-90%) that are unprecedented, especially in relapsed and refractory acute B cell leukemia.Nonetheless, the field of CAR T cells is currently facing two major challenges. First, although CAR T cells can be extremely effective in killing malignant cells, they can also cause deleterious side effects, including off-tumor toxicity and cytokine release syndrome.Second, the CAR T cell strategy has not yet produced favorable clinical responses in targeting solid malignancies.Up to now, most clinical trials on CAR T cells in solid tumors indicate no or scant objective responses (see Fuca et al.for a comprehensive presentation of clinical trials on solid tumors). This is, for example, the case of sarcoma patients infused with human epidermal growth factor receptor (HER)-2 CAR T cells,or pancreatic carcinoma patients infused with mesothelin CAR-T cells.Most worrisomely, severe toxicities caused by on-target but off-tumor antigen recognition by CAR T cells have been reported. In one study, the infusion of CAR T cells targeting HER-2 caused fatal acute respiratory distress syndrome due to recognition of lung epithelia cells expressing low levels of HER-2. ## T cell homing and migration in the context of car t cell therapy ## Principles of adoptive t cell therapy ## Homing, migration, and persistence of therapeutic t cells in target tissues As stated earlier, CAR T cells have shown remarkable success in B cell malignancies. Many of these semiliquid hematologic malignancies reside in the bone marrow, a niche easily accessible for intravascular T cells. In solid tumors, the situation is strikingly different and CAR T cells face additional barriers that lymphocytes must break in order to control tumor growth . First, CAR T cells need to home into the tumor during the extravasation phase by crossing tumor blood vessels. A study performed in a metastatic breast cancer patient with HER-2-specific T cells demonstrated the incapacity of infused lymphocytes to home into solid tumor masses. This is in sharp contrast with the accumulation of tumor-specific T cells into the cancer patient's bone marrow.Similar results obtained in preclinical mouse tumor models led to the conclusion that T cell homing into solid tumors is a limited process.Key factors for T cell entry into tumor masses are chemokine receptors and adhesion molecules. We know that active trafficking of T cells into tumors partially depends upon the compatibility between chemokines found in tumor and chemokine receptors expressed on T cells. Once T cells enter the tumor, they must disseminate and physically contact their targets . In carcinomas, which represent 90% of solid tumors, tumor cells are organized in islets surrounded by specialized microenvironment, referred to as the stroma. As tumor blood vessels are localized in the stroma, newly entered T cells must migrate from their entry points to their targets. This interstitial migration is influenced by many external elements such as soluble factors as well as by cellular and structural determinants. ## Car t cells migrating towards healthy tissues car t cell recruitment from the blood car t cell migration in the tumor ## F i g u r e 1 homing, migration and persistence of chimeric antigen receptor (car) t cells in tumors. CAR T cell therapy comprises T cell isolation followed by a CAR transduction and in vitro expansion of the CAR T cells. After the T lymphocytes are reinfused, they need to be recruited from the blood to the tumor site. There, the engineered receptors are able to interact with specific antigens present on tumor cells and, ultimately allowing the cytotoxic killing. Additionally, persistence of CAR T cells either in the tumor site or in the peripheral blood is critical for robust clinical responses Few studies have documented the navigation of T cells within the tumor stroma. By using a model of fresh human tumor slices combined with dynamic imaging microscopy, we have monitored the motility of resident T cells in human solid tumors. Overall, we found that T cells migrate poorly in the stroma of human lung tumors (2.5 m/min of mean velocity from 9 patients) highlighting the presence of obstacles that impede T cells from reaching cancer cells.This notion is reinforced as, in few patients whose tumor islets are enriched in T cells, lymphocytes move fast in the stroma. After their navigations within the stroma, T cells need to form productive conjugates with their targets . Imaging experiments have provided insights in the way T cells engage malignant cells. At the molecular levels, we know that together with the binding of the TCR with peptide-MHC complexes, adhesion molecules are important.Among these, two integrins, namely LFA-1 and E 7, play a critical role in T cell/tumor cell interaction. LFA-1 binds to ICAM-1 (CD54) expressed by antigen-presenting cells as well as by some tumor cells.When T cells are stimulated through their TCRs or chemokine receptors, the affinity and clustering of LFA-1 increases in an inside-out signaling process that promotes the binding of this integrin to ICAM-1. Whether similar molecules and mechanisms control the antitumoral action of CAR T cells remains to be established. ## In vitro and in vivo experiments have also documented the kinetic interactions between t cells, including car t cells and tumors cells. In vitro experiments performed with murine CAR T cells indicate that lymphocytes sequentially contact and kill several tumor cells, with faster formation of a synapse and more rapid detachment of tumor cells compared with TCR-mediated cytotoxicity.In vivo experiments support the notion of fast killing dynamics for CAR T cells, which would contribute to their therapeutic efficacy. Indeed, CD19 CAR T cells were observed to engage, kill, and detach from malignant B cells in ∼25 min.This rapid detachment that would favor serial killing contrasts with previous observations of conventional CD8 + T cells forming long-lasting interactions with tumor cells. Finally, to successfully control the tumor, CAR T cells need to persist for a long time in cancer patients . In patients with hematologic malignancies, it has been shown that the persistence of CAR T cells in peripheral blood is an essential factor for durable clinical response. It is likely that the same holds true for solid tumors, although no evidence is available for the moment. The determinants that control T cell persistence are actively searched and linked to epigenetic factors controlling the lymphocyte differentiation process.In particular, the process of T cell exhaustion associated with chronic antigenic activation and specific epigenetic marks may account for the restricted efficacy of CAR T cells. As recently reported, overexpression of the transcription factor JUN in CAR T cells is a promising strategy to counteract their exhaustion and improve their therapeutic efficacy.Optimally, CAR T cell therapy should combine the activity of cells with immediate cytolytic effector function to kill the bulk of fastgrowing tumors and the persistence of tumor-specific cells with selfrenewal capabilities to provide a prolonged supply of cytolytic effector progeny to ensure long-term control over tumor cells. Current in vitro methods employed to expand cells to sufficient numbers and still maintain a minimally differentiated phenotype are hindered by the biologic coupling of clonal expansion and effector differentiation.Therefore, a better understanding of the physiologic mechanism that couples cell expansion and differentiation of CD8 + T cells may improve the efficacy of T cell based immunotherapy. ## Barriers to t cell motility in the context of immunotherapy ## Defects in t cell entry The endothelium is a key checkpoint to control leukocyte recruitment during an inflammatory response. Particularly, T cells will have to cross the vasculature in order to access the target tissue. A number of factors are involved in this step, including chemokines and adhesion molecules responsible for the tethering, rolling, adhesions and transmigration phases through the activated endothelium. Aberrant vasculature operates as a first hindrance for T cell trafficking into the tumor sitesleft panel). Tumor endothelium is regarded as structurally immature and disorganized, with a critical role for the higher activity of proangiogenic factors, which is associated to abnormal blood flow and permeability, ultimately leading to increased tissue pressure, collapse of the blood vessels, and dysregulated oxygen supply.For CAR T cells, a recent study using a positron emission tomography probe to assess lymphocyte trafficking did find the presence of intravenously injected engineered cells in mouse bearing tumors positive for the disialoganglioside GD2.However, the accumulation of CAR T cells into tumors was relatively slow, peaking 2 wk after the initial transfer. This access problem is even magnified when dealing with brain tumors, forcing investigators to circumvent the blood-brain barrier by intracranial injection. ## Defect in t cell migration within tumors In many tumors, although lymphocytes have successfully crossed tumor blood vessels, T cells are not in contact with malignant cells but instead enriched in the surrounding stroma.This so-called "excluded immune infiltrate" phenotype is associated with a lack of responsiveness to PD-1 blockade, stressing the importance of stromal obstacles that need to be identified in order to overcome them. The reasons for this T cell sequestration in the tumor stroma are manifold, as pointed out below. ## Extracellular matrix (ecm) barrier Increase in tumor stiffness is a well-established feature of growing tumors. This is due to an increased deposition and aberrant archi- ## Hypoxia Hypoxia, a feature of growing tumors, has been shown to dampen T cell functions. ## Chemokines Chemokines, a group of cytokines with chemotactic activity, have been tumor islets by a mechanism, which remains to be clarified. ## Defect in synapse formation between t cell and malignant cells Once T cells have passed the stromal obstacles described earlier, theyThese observations led to the conclusion that CARs, although of high affinity for their targets, are not very sensitive and require a high amount of antigen at the cancer cell surface. This is in sharp contrast with the functioning of TCRs, which are highly sensitive receptors able to recognize a very limited numbers of peptide-MHC complexes.The molecular mechanisms underlying the low CAR sensitivity is actively searched with attention paid to the signaling elements of the chimeric receptor. Hence, a recent study has demonstrated that for low antigen densities, CARs with CD28 signaling domains were much more potent than CARs with 4-1BB signaling domains.Moreover, strategies aiming at increasing the surface expression of the antigen targeted by the CAR have been recently implemented to prevent resistance to CAR T cell therapy.Altogether, the notion that emerges from these findings is that progressing tumors have accumulated multiple mechanisms impeding T cells from migrating and contacting tumor cells. ## Car t cell aberrant migration Apart from a defective migration into and within the tumor, effector T cells can also exhibit aberrant distribution, which can lead to severe toxicities. Cytokine-release syndrome and CAR T cell-related encephalopathy syndrome are the two most-common life-threatening toxicities observed after CAR T cell therapy, although rare cases of anaphylaxis have also been reported.The pathophysiologic mechanism underlying these toxicities remains to be determined. Nevertheless, accumulating evidence suggests that neurotoxicities are associated with a disruption of the blood-brain barrier and the infiltration of CAR T cells into the CNS. ## Rewiring t cell motility as an opportunity to improve immunotherapy Along its journey toward a specific tumor target, a T cell will face complex interactions with distinct tissue barriers, such as blood vessels, the tumor microenvironment, as well as the tumor tissue itself. Thus, directly targeting molecular components involved in T cell adhesive and motility activities, might overcome tissue obstacles for efficient trafficking and lead to improvement of CAR T cell therapy, middle panel). ## Targeting tumor vasculature to improve t cell migration An abnormal expression of angiogenic growth factors is a hallmark of growing tumors. Therefore, anti-angiogenic strategies aimed at normalizing the structure of tumor vasculature, and a likely subsequent reversal to an increased expression of endothelial adhesion molecules, might decisively enhance T cell recruitment and response to tumor tissues.In fact, in a murine melanoma model, disruption of vascular endothelial growth factor/VEGF receptor-2 (VEGF/VEGFR-2) signaling by treatment with anti-VEGF showed a synergistic efficiency with an immunotherapy employing adoptively transferred T cells carrying a transgenic TCR directed to a melanoma antigen, resulting in enhanced tumor tissue infiltration.Moreover, in another murine cancer model, combined neutralization of VEGF/angiopoietin-2 led to improved tumor control, as a result of both better response with anti-PD-1 blockade and increased accumulation of perivascular CD8 + T cells.As targeting of VEGF also disrupts its immunosupressive effects on the tumor microenvironment, such a strategy deserves further investigation as a countermeasure to the tumor microenvironment devoid of infiltrating T cells.Additionally, this approach can also be part of a thoughtful multistrategic therapy for combating cancer. The combination of angiogenic blockade and immunotherapy along with radiotherapy, which also leads to overexpression of adhesion molecules in the target tumor vessels, might represent an effective pathway to enable T cells to access the tumor site. ## Targeting cells and extracellular components in tumor microenvironment to improve t cell migration The tumor microenvironment seems to be a dysregulated battlefield where recruited leukocytes contribute distinctly for antitumoral immunity or for a pro-tumorigenic microenvironment via modulation of the specific immune response. In fact, an immunosuppressive outcome follows the presence of metabolic alterations and the secretion of immunoregulatory cytokines. Besides, suppressive activities are also exerted by distinct subpopulations infiltrating the tumor site, such as Treg cells and myeloid cells.All these components play a role in limiting the activity of CAR T cells and their subsequent capability to gain access to the tumor tissue. Therefore, controlling the activity of immunosuppressive components associated to the tumor site seems a relevant strategy aimed at improving CAR T cell therapy. In this context, Treg depletion has been regarded as an important mechanism of anti-CTLA-4 immunotherapy.Interestingly, an experimental study showed a significant increase in CD8 + T cell infiltration and tumor rejection following extensive Treg depletion, a finding that might be associated to tumor vasculature normalization, which express increased levels of VCAM-1 and ICAM-1 on endothelial cells.Moreover, as recent reports have pointed, other particular cell subsets act as critical players in avoiding intratumoral T cell infiltration. These negative regulators probably affect CAR T cells too, although further studies on specific cell depletion to improve CAR T cell trafficking are warranted. As stated previously, macrophages have a detrimental impact on T cell ability to reach cancer cells, supporting the use of strategies that either deplete or reprogram macrophages. Hence, in several mouse tumor models the depletion of macrophages allowed CD8 + T cells to infiltrate and mediate tumor regression when combined with anti-PD-1 blockade.This aspect takes an increasing importance, as macrophages have also been associated with some forms of toxicity (e.g., cytokine release syndrome) after CAR T cell infusion.However, one must keep in mind that macrophages with a proinflammatory phenotype can also exert antitumoral activities either alone or in cooperation with T cells, stressing the risk of a depletion strategy.Approaches to target cancer-associated fibroblasts, which are responsible for the excessive production of the ECM limiting intratumoral T cell migration, have been also implemented. In a lung cancer model, a CAR T cell was engineered to successfully deplete cancer-associated fibroblasts, which comprise a central cellular subset of the tumor microenvironment.As expected, such a strategy inhibited tumor growth even more significantly by co-targeting cancerassociated fibroblasts and the specific tumor antigen. Although the mechanistic basis for this enhanced antitumor activity was not provided, the effective T cell infiltration might be related to normalization in tumor vascularization, mitigation of an immunosuppressive milieu, as well as modulation of the ECM content. Other strategies aim at directly targeting the ECM, because CAR T cells have a limited capability to degrade it and, therefore, might harbor an impaired trafficking toward the tumor tissue. In fact, in vitro expansion of CAR T cells leads to down-regulation of HPSE gene and a consequent loss of heparanase, an enzyme that T cells secrete to degrade heparan sulfate proteoglycans in ECM.By engineering CAR T cells to coexpress heparanase, these cells showed both an improvement in ECM degradation and an increase of in vitro migration activity, as ascertained by a Matrigel-based cell invasion assay. Moreover, enhanced tumor infiltration and decreased tumor growth correlated with an improved mouse survival. The ECM as a pivotal component of the CAR T cell-resistant tumor stroma could also be evidenced from the striking observation that CAR T cells specific for chondroitin sulfate proteoglycan 4 (a melanoma surface proteoglycan) demonstrated a relevant antimelanoma activity, whereas co-targeting strategy of heparan sulfate proteoglycans and CD19 did not seem to be efficient to control the stroma-poor B cell lymphoma.The association of classical chemotherapy or radiotherapy strategies to the CAR T cell therapy has been postulated to play a role in targeting the tumor microenvironment, with a potential impact on CAR T cell trafficking. Previously, the effect of ablative radiotherapy upon primary tumor and metastasis was reported to be strongly dependent on CD8 + T cell response.Radiotherapy seems to induce an inflammatory milieu, which might favor increased vascular adhesion and chemotaxis-driven migration.In addition, irradiation of cancer cell lines led to increased expression of tumor-associated antigens.Therefore, an efficient CAR T cell migratory and infiltration capabilities might benefit from a combination to a classical radiotherapy to treat solid tumors. ## Targeting chemotactic response to improve t cell migration Targeting of chemokine-chemokine receptor signaling has been tested in several preclinical and clinical studies. Particularly, T cell trafficking into tumor sites following endothelial transmigration is also governed by a proper response to the chemokine milieu in these sites. In this sense, as effector memory T cells bear high densities of CXCR3 and CCR5 chemokine receptors, it is expected that they are able to infiltrate and target tumors producing chemokines that signal through these receptors. In fact, CCR5 was the first chemokine receptor demonstrated to be involved in the infiltration of cytotoxic T cells to tumor site.Such a finding could further evolve as a genetic approach to tune the migratory activity of T cells, in order to redirect them toward a given chemokine secreted by tumor cells.tumors. An approach employing CAR T cells along with an oncolytic vaccinia virus engineered to produce CXCL11, a CXCR3 ligand, showed higher efficiency in recruiting CAR T cells and control tumor progression in comparison to the viral therapy alone. Moreover, the authors also demonstrated comparatively that an approach by engineering those CAR T cells to produce CXCL11 was not able to increase T cell migration into tumoral site, despite the increased local chemokine production.Additionally, immunosuppressive factors, such as PGE 2 and adenosine, are reported to be enriched in the tumor microenvironment of solid tumors and exert potent inhibition of T cell functions, including migration and adhesion. As this inhibition is dependent on cAMPdependent protein kinase A (PKA) activation and its recruitment to the immune synapse by anchoring to the membrane protein ezrin, an interesting approach by targeting intracellular determinants of such an effect was envisaged to improve CAR T cell activity.Blockage of PKA-ezrin interaction, by employing the "regulatory subunit I anchoring disruptor" (RIAD), restricted the cAMP/PKA-mediated immunosuppression and the engineered CAR-RIAD T cells carry increased antitumor activity in vivo, when compared to CAR T cells. Noteworthy, CAR-RIAD T cells also showed higher baseline expression of CXCR3 and the adhesion molecule CD49d, a finding that might be, respectively, correlated with their increased CXCL10-driven chemotaxis and adhesion to fibronectin and VCAM-1.This report is pivotal in highlighting intracellular determinants of T cell function as potential targets to engineer enhanced efficacy for CAR T cell therapy. Besides these modulatory strategies on chemotaxis, it is important to point out the regional delivery of CAR T cells as a potential approach to surmount the obstacles for T cell migration toward the target tumor.By increasing the efficacy of CAR T cells to target solid tumors, such an approach might also limit their off-tumor toxicity. In fact, both experimental and clinical studies indicate effective action of locally delivered CAR T cells against brain tumors. 107-110 ## Importance of preclinical models in assessing car t cell dynamics One of the key challenges in the field of cancer immunotherapy, including CAR T cells, is to develop relevant models to test and predict the efficacy and safety of cell products.As stated earlier in our review, migration largely contributes to the efficacy of CAR T cells but can also lead to toxicity, underlining the need of relevant models to track engineered lymphocytes. Historically, in vitro approaches and mouse tumor models have been extensively used to study CAR T cell migration. Cell culture methods typically rely on cancer cells or immortalized cells grown within artificial environments admixed with CAR T cells. Although in vitro cell culture models do not integrate the complexity of the tumor environment, they possess a number of advantages. In particular, they are easily amenable to imaging microscopy and, in this context, several reports described the dynamics of CAR T cells during their contact with tumor cell lines.It is expected that such models combined with high-resolution microscopy will particularly be useful to provide insights on the structure of the immune synapse. Efficacy of CAR T cells is usually tested in mice harboring tumors. to monitor lymphocyte bio-distribution noninvasively using clinical imaging modalities (e.g., with positron emission tomography reporter gene).A key limitation of these xenografted models is a lack of immune cells and components that presumably take an important part in controlling the trafficking of CAR T cells. Recently, there has been a marked effort to develop more relevant mouse models to test immunotherapy products. In this regard, humanized mice harboring tumors have been successfully used to evaluate CAR T cell toxicity.In addition to xenografted models, studies in immune-competent mice with murine CAR T cells have been conducted. Combined with intravital two-photon microscopy techniques, they have unraveled the behavior of CD19 CAR T cells in time and space.Nevertheless, although these models provide very valuable information, they do not always recapitulate human biology. For example, the murine immune system, including its composition and phenotype, differs from that in humans. This is important when one deals with toxicities associated with CAR T cells, which have been largely missed in immunocompetent mouse models.Beyond murine models, embryonic zebrafish xenografts have recently been proposed as an alternative for preclinical evaluation of CAR T cells.Zebrafish embryos are permissive to human cell transfer including tumor cells and T cells. Because of their size and transparency, they appear particularly suited for tracking the motility and killing activities of CAR T cells with high resolution and high throughput. Additionally, 3D models, including organoids, are increasingly popular these days. They usually consist of purifying cancer cells from a fresh human biopsy, followed by an aggregation step. Their use in testing immunotherapy reagents, including CAR T cells, is fast-growing.They are patient specific, easily cultured several weeks and amenable to dynamic imaging microscopy. However, they usually lack the structure observed in human carcinomas with compact tumor islets surrounded by a stroma. Traditionally established by neurobiologists to study neuronal electrical activities in brain slices, organotypic models have been implemented to monitor the dynamics of immune cells, first in lymphoid organs and then in tumors.Although the structure of the tumor is mostly preserved, one drawback is the difficulty to keep tumor explants several days in culture limiting the range of possibilities. We believe that all these issues and questions will be approached through the development of proper and relevant preclinical models, gene editing technologies, such as CRISPR-Cas9 method,and innovative imaging technologies. ## Concluding remarks ## Authorship All authors contributed to both the review conceptualization and the writing process. # Acknowledgments The main guide for writing this review have been the stimulating discussions gathered during the previous editions of the leukocyte motility course "LymphoRioMove". We wish to thank the participating stu- # Disclosures The authors declare no conflicts of interest. ## Orcid Loïc Dupré https://orcid.org/0000-0002-7278-6503 Vinicius Cotta-de-Almeida https://orcid.org/0000-0002-6753-3480

The social contagion of temporal discounting in small social networks Decisions often require a tradeoff between immediate and long-term gratification. How individuals resolve such tradeoffs reflects constructs such as temporal discounting, the degree that individuals devalue delayed rewards. Recent research has started to focus on temporal decisions made in collaborative contexts (e.g., dyads, small groups). Results suggest that directly interacting with others leads to revisions in preferences, such that decision makers become more similar to their collaborative partners over time (e.g., more patient following collaboration with a patient other). What remains to be seen is whether this social influence extends to indirect social effects, such as when an individual influences another's preferences through a shared collaborative partner. In the current study, the focus was on decisions regarding hypothetical monetary rewards. Groups of three participated in a collaborative decision-making chain, in which network member X collaborated with member Y, who then subsequently collaborated with member Z. Though network members X and Z never directly interacted, a significant indirect link was observed between member X's pre-collaborative decision preferences and member Z's post-collaborative decision preferences. These results demonstrate that temporal decision preferences can be transmitted through intervening connections in a small social network (i.e., social contagion), showing that indirect social influence can be empirically observed and measured in controlled environments. # Introduction Many beneficial outcomes require a tradeoff between short-term and long-term gratification (e.g., good health, retirement savings). Resolving such tradeoffs reflects psychological constructs such as temporal discounting-the extent to which an individual prefers more immediate rewards over delayed rewards. Temporal discounting relates to many consequential behaviors, including addiction [bib_ref] Behavioral and neuroeconomics of drug addiction: Competing neural systems and temporal discounting..., Bickel [/bib_ref] , health outcomes [bib_ref] Steep discounting of delayed monetary and food rewards in obesity: A metaanalysis, Amlung [/bib_ref] , and suicidality [bib_ref] Lethal forethought: Delayed reward discounting differentiates high-and low-lethality suicide attempts in old..., Dombrovski [/bib_ref]. Therefore, a better understanding of how temporal preferences are formed and modified will have practical benefits for a variety of behavioral outcomes, particularly for interventions that target high rates of temporal discounting (e.g., [bib_ref] Change in delay discounting and substance reward value following a brief alcohol..., Dennhardt [/bib_ref]. Less is known about the degree to which temporal decision preferences are socially influenced. Humans exist within social networks, with their behaviors and preferences influenced by their social contacts (referred to as social contagion;. Social contagion has been observed in variables that involve the tradeoff between short-term and long-term consequences, such as smoking behavior (Christakis and Fowler 2008) and body-mass index [bib_ref] The spread of obesity in a large social network over 32 years, Christakis [/bib_ref]. Taken together, it might be expected that temporal decision preferences themselves exhibit strong social influence effects. However, most studies that looked at network effects have been observational studies, which make it difficult to isolate causal social influence. The current study aimed to help fill this gap in the literature by studying social influence on temporal decision making in a controlled environment. First, it is important to distinguish between two types of social effects: direct and indirect influence. ## Direct social influence Direct social influence in a social network refers to the influence of one individual on another individual with whom they come into direct contact. Common examples include friends, family members, and work colleagues. Direct influence can stem from behavioral imitation, informational effects, and conformity to group norms. As it relates to temporal decision making, most research has focused on individual effects, making the role of social influence less clear. Recent research has demonstrated that observing the choices of a peer over the computer influenced intertemporal choices in a sample of young adults [bib_ref] Impulsive social influence increases impulsive choices on a temporal discounting task in..., Gilman [/bib_ref]. For example, observing an impulsive peer led to a greater preference for smaller-sooner monetary rewards over larger-later rewards. Another way to test direct social influence is to have a group of participants interact together in a laboratory setting [bib_ref] Let's decide together: Differences between individual and joint delay discounting, Schweke [/bib_ref] [bib_ref] How are individual time preferences aggregated in groups? A laboratory experiment on..., Tsuruta [/bib_ref]. In the collaborative decision-making paradigm (Bixter and Rogers 2019; [bib_ref] Are intertemporal preferences contagious? Evidence from collaborative decision making, Bixter [/bib_ref] , dyads or small groups of participants make decisions together during a collaboration phase. Because participants also complete the decision-making task individually both precollaboratively and post-collaboratively, direct social influence can be assessed by measuring the extent to which an individual's preferences are related to how their partner's preferences change from pre-collaboration to post-collaboration. That is, does collaborating with another person lead to revisions in decision preferences? Results from this paradigm demonstrate that the temporal decision preferences of group members are revised to be more similar post-collaboratively than they were pre-collaboratively. The extent of this direct social influence depends on the relative status that someone has within the group, with lower-status members exhibiting larger social influence effects than higher-status members [bib_ref] Delay discounting in dyads and small groups: Group leadership, status information, and..., Bixter [/bib_ref]. ## Indirect social influence The social contagion of behavior ultimately relies on the spread of behavior through social networks, including between individuals who never directly interact with one another. An example of an indirect link is if individual X and individual Z never interact with each other, but both interact with individual Y. Therefore, indirect social influence refers to the influence of one network member on another member with whom they do not come into direct contact. Though direct social influence has been observed and measured in the social psychological literature for nearly a century [bib_ref] Studies of independence and conformity: I. A minority of one against a..., Asch [/bib_ref] , indirect social influence is a relatively recent topic of study and permits the results to be more easily scaled to the complex structures and characteristics of social networks. Most research that has focused on social network effects has used observational methods (e.g., [bib_ref] Exercise contagion in a global social network, Aral [/bib_ref]. In certain situations, behavior is not just influenced by direct contacts, but also by contacts separated by two degrees or more of separation (i.e., indirect links; [bib_ref] Alone in the crowd: The structure and spread of loneliness in a..., Cacioppo [/bib_ref] [bib_ref] The spread of alcohol consumption behavior in a large social network, Rosenquist [/bib_ref]. These results suggest that behaviors can spread throughout social networks similar to contagious diseases. Observational social network studies have not gone without criticism, though, mainly due to them having difficulty separating causal social influence from other effects such as homophily [bib_ref] Homophily and contagion are generically confounded in observational social network studies, Shalizi [/bib_ref] [bib_ref] Is obesity contagious? Social networks vs. environmental factors in the obesity epidemic, Cohen-Cole [/bib_ref]. Homophily refers to the tendency of people to choose their network connections based on shared characteristics (also referred to as selection effects). For instance, if clusters of individuals in social networks form their ties based on shared characteristics (e.g., smoking status, exercise habits), similarities observed between proximal network members might not be due to social influence. Context effects refer to environmental variables that may affect individuals in close proximity to one another (and are, thus, more likely to be connected within the larger social network). An example would be if a new fast food restaurant opens up next to an office building, which then leads to many of the workers in the office building (who share many direct connections) seeing their body-mass index scores increase over time. Controlling the environment in laboratory settings can better isolate indirect social influence and information cascades more generally [bib_ref] Distinguishing informational cascades from herd behavior in the laboratory, Çelen [/bib_ref] [bib_ref] Information cascades in the laboratory, Anderson [/bib_ref]. One area that has received particular attention is the spread of generosity, cooperation, and other pro-social behaviors (e.g., [bib_ref] Contagion of cooperation in static and fluid social networks, Jordan [/bib_ref] [bib_ref] Direct and indirect influence of altruistic behavior in a social network, Liu [/bib_ref] [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref]. The findings regarding social influence on altruistic behavior have been mixed, with some studies finding significant indirect social influence and others not [bib_ref] Cooperation and contagion in web-based, networked public goods experiments, Suri [/bib_ref]. These experimental studies demonstrate that studying social networks in controlled environments helps to make clear the scope of indirect influence on decision behavior. For instance, even though direct social influence may be observed in a decision domain, social influence may not extend to indirect connections (e.g., [bib_ref] Direct and indirect influence of altruistic behavior in a social network, Liu [/bib_ref]. In the context of temporal decision making, no laboratory study to date has measured whether decision preferences can be transmitted indirectly throughout small social networks. By separating the discussion of direct and indirect social influence, we are not claiming that these two types of influences are fundamentally separate phenomena. After all, indirect social influence is just an aggregation of constituent direct social effects-if an indirect social effect links members X and Z through a shared link with member Y, this indirect influence derives from direct effects between members X and Y and members Y and Z. However, as the research above demonstrated, results are mixed on the presence of indirect social influence in decision making. As a result, just because direct social influence has been observed in a domain of interest such as temporal decision making, it does not necessitate that this influence will extend to indirect links within a social network. The presence or absence of indirect social effects places boundaries on the impact that social influence is likely to have in real social networks, making it a prime target of behavioral research. ## Overview of current study To test both direct and indirect social influence on temporal decision making in a controlled, experimental environment, a sequential chain design was chosen. Small social networks consisting of three members (X, Y, Z) were included in each study session. All three network members first completed a temporal decision-making task individually to assess their baseline, pre-collaborative decision preferences regarding delayed rewards. Network members X and Y then collaborated together face-to-face as a dyad, where they completed the temporal decisionmaking task together. Subsequently, network members Y and Z collaborated together as a decision-making dyad. Finally, all three network members again completed the decision-making task individually to assess their postcollaborative decision preferences (see for a visualization of the study sequence). Due to the sequential chain structure of the study design, network members X and Z only shared an indirect link through participant Y. Therefore, any indirect relationship between member X's pre-collaborative preferences and member Z's post-collaborative preferences would demonstrate the social contagion of temporal decision making. Furthermore, due to the ordering of the sequence chain, influence between network members X and Z could only flow from X to Z, not vice versa. This study design allowed a more definitive test of indirect social influence compared to previous observational studies. The focus on temporal decision making was due to intertemporal tradeoffs underlying many everyday decision conflicts. The particular task used in the current A visualiazation of the full path model fit to the small-network chain sequence of the current study. Three network members (X, Y, Z) completed a temporal decision-making task individually (Ind) and in collaborative dyads. The network members completed the individual conditions both prior to collaboration (Pre) and after collaboration (Post). The path lettering is used to help identify the various direct and indirect social effects included in Tables 1 and 2. Paths included in the model but not shown in the figure for illustration purposes include: direct paths from network members' pre-collaborative discount rates to their respective post-collaborative discount rates (e.g., Ind X Pre → Ind X Post), covariances between network members' pre-collaborative discount rates (e.g., Ind X Pre ↔ Ind Y Pre), and covariances between post-collaborative residuals (e.g., Ind X Post ↔ Ind Y Post). The latter covariances were included to take into account the dependency of the group data study dealt with hypothetical monetary rewards. Previous studies have often found no significant differences between temporal discounting tasks that used real vs. hypothetical monetary rewards [bib_ref] Within-subject comparison of real and hypothetical money rewards in delay discounting, Johnson [/bib_ref] [bib_ref] Delay discounting of real and hypothetical rewards III: Steady-state assessments, forced-choice trials,..., Lagorio [/bib_ref]. As a result, the use of hypothetical monetary rewards was deemed acceptable for this initial investigation of social network effects on temporal discounting. However, we describe in the "Discussion" section how future research can build upon the current study by utilizing a variety of incentivized and real-world decision tasks. # Method ## Participants Participants were 117 undergraduate students, consisting of 39 small social networks of three. The mean age of the sample was 19.7 years (SD = 2.40) and 54.7% were female. Participants received partial course credit for completing the study. The entire study took less than 1 h to complete. # Materials ## Temporal decision-making task The decision-making task involved participants making decisions about hypothetical monetary rewards. On each trial, two rewards were presented on the screen. One of the two reward's magnitude was missing (e.g., US$125 today or US$___ in 6 months). Participants' task was to respond with the missing reward magnitude that would render them indifferent between the two rewards. Each implementation of the decision-making task consisted of 36 trials, with the order of the trials randomized. The 36 trials derived from three reward magnitudes (US$40, US$125, and US$250), three delays (3 months, 6 months, and 12 months), whether the sooner reward would be delivered today or also after a delay, and whether participants had to provide a missing value for the sooner reward or the later reward. The task was either completed individually or in a collaborative dyad, where the two network members would have to reach consensus on each trial. ## Discount rates Responses on each trial were converted to annual discount rates using Eq. 1 [bib_ref] Discounting time and time discounting: Subjective time perception and intertemporal preferences, Zauberman [/bib_ref] where X t is the magnitude of the smaller-sooner reward, X t + h is the magnitude of the larger-later reward, t is the delay associated with the smaller-sooner reward, and h is the additional delay (in months) associated with the (1) Annual discount rate = ln X t+h X t h 12 , larger-later reward. Higher discount rates imply greater devaluing of delayed rewards (i.e., greater impatience). The denominator involves dividing h by 12 to express the discount rate as an annual discount rate. For instance, if a participant replied US$150 in 6 months to the example trial above, the annual discount rate for that trial would be 0.36. However, if the participant required the reward to be US$500 in 6 months, the participant's annual discount rate would be much higher at 2.77. Discount rates were calculated for each individual participant and each dyad by computing the discount rates implied by each response and then averaging the resulting set of discount rates across the 36 trials. The overall average discount rate across all network members and phases was 2.55 (SD = 1.03). ## Procedure The design of the study can be seen in . Three participants (X, Y, Z) were included in a single study session as a small-network chain. Participants initially arrived and were placed into different testing rooms to minimize face-to-face interaction prior to any collaboration. Participants were not aware of the network design of the study or their location within the chain structure. The study was broken down into the following four phases (see . Phase 1 Network members X and Y completed the temporal decision-making task as individuals (Ind X Pre and Ind Y Pre). They completed the task in separate rooms and were unaware that they would subsequently be completing a similar task in a collaborative context. These individual pre-collaborative tasks acted as baseline measures of the network members' temporal decision preferences. Phase 2 After completing their individual pre-collaborative tasks, members X and Y collaborated as a dyad (Dyad XY). During the collaborative task, the dyad members were asked to reach a consensual decision on each trial. As this collaborative task was being completed by members X and Y, the third member of the chain, participant Z, completed the task individually in a separate room (Ind Z Pre). Members X and Z never came into face-to-face contact to help to ensure that their indirect link was uncontaminated. Phase 3 The first member of the chain, participant X, completed another individual phase of the task (Ind X Post). This phase acted as a post-collaborative measure of their temporal decision preferences following their direct interaction with participant Y. Also, during Phase 3, network members Y and Z collaborated together as a second dyad (Dyad YZ). Phase 4 Network members Y and Z once again completed the temporal decision-making task individually (Ind Y Post, Ind Z Post). These individual tasks measured the two members' post-collaborative decision preferences. The sequential chain structure of the current study allowed both direct and indirect social influence to be measured within a single design and model. Direct effects could be assessed by the joint, multiplicative pathways between pre-collaborative and post-collaborative decision preferences of network members who directly interacted with one another. For example, did the precollaborative decision preferences of network member Y influence the post-collaborative decision preferences of member X following the Dyad XY experience? Moreover, because network members X and Z never directly interacted with one another, indirect social influence from X's pre-collaborative decision preferences (Ind X Pre) to Z's post-collaborative preferences (Ind Z Post) would demonstrate that temporal decision preferences can cascade and propagate throughout small social networks. # Analysis plan The path model displayed in was fit to the data. This analytic approach allowed direct and indirect social influence to be estimated within a single model, as well as taking into account the dependency of the group data. Additional paths were estimated in the model but were not shown in for illustration purposes, including direct paths from network members' precollaborative decision making to their respective postcollaborative decision making (Ind X Pre → Ind X Post, Ind Y Pre → Ind Y Post, Ind Z Pre → Ind Z Post). These autoregressive path coefficients adjust network members' post-collaborative discount rates by their respective precollaborative discount rates, so that any unique influences of the dyad interactions can be seen as influences on the change in network members' discount rates. Covariances were also estimated between network members' precollaborative decision making (Ind X Pre ↔ Ind Y Pre, Ind X Pre ↔ Ind Z Pre, Ind Y Pre ↔ Ind Z Pre), as well as covariances between the post-collaborative residuals (Ind X Post ↔ Ind Y Post, Ind X Post ↔ Ind Z Post, Ind Y Post ↔ Ind Z Post). These covariances were included to take into account the dependency of the group data. The path models used maximum likelihood estimation with bootstrapped standard errors (based on 10,000 resamples). Bias-corrected 95% confidence intervals were used to test the significance of all path coefficients (including the indirect effects) of the models [bib_ref] Confidence limits for the indirect effect: Distribution of the product and resampling..., Mackinnon [/bib_ref] ; if the confidence limits of an interval did not include zero, the coefficient was deemed statistically significant. # Results Path coefficients derived from the path model in are included in . We first report the various direct social effects in the model, followed by the indirect social Coefficient estimates of the path model displayed in Paths labeled as "-" were not included in for illustration purposes. CL confidence limit. Confidence limits were derived from 95% bias-corrected bootstrap confidience intervals based on 10,000 resamples. The significance of the path coefficients ( a ) was based on whether the confidence interval included zero or not . Reported coefficients are the unstandardized estimates followed by the 95% bias-corrected confidence intervals in square brackets. ## Direct social influence The three-member chain structure of the current study allowed a variety of direct social effects to be estimated. The clearest test of a direct effect focuses on the first member of the chain, member X. This member only has a direct interaction with one other network member (member Y), and their individual decision preferences were assessed immediately before and after this collaboration. The multiplicative pathway from member Y's precollaborative discount rates (Ind Y Pre) to member X's post-collaborative discount rates (Ind X Post), through the intervening dyadic collaboration (Dyad XY), was significant . This effect suggests that member Y exerted a significant direct social influence on member X's revised preferences. That is, member Y's baseline preferences had a unique effect on the decision preferences exhibited by the XY dyad during collaboration, which then uniquely predicted member X's postcollaborative preferences. Because autoregressive paths were included in the path model (e.g., Ind X Pre → Ind X Post), any unique influence of the Dyad XY collaboration is on member X's post-collaborative preferences adjusting for their baseline preferences. To further investigate this direct social effect, a multiple regression was performed with member X and Y's pre-collaborative discount rates (Ind X Pre, Ind Y Pre) predicting member X's post-collaborative discount rates (Ind X Post). This model more clearly demonstrates the influence of member Y on member X by measuring the direct unique effect of member Y's baseline preferences on member X's revised, post-collaborative preferences. Both Ind X Pre (B = 0.55, SE = 0.11, P < 0.001) and Ind Y Pre (B = 0.42, SE = 0.13, P = 0.003) were significant unique predictors of Ind X Post, F(2, 36) = 21.63, P < 0.001, R 2 = 0.55. These results demonstrate that network member X's preferences were revised following the collaboration with member Y, so that their postcollaborative discount rates were a combination of their baseline preferences and the preferences of their collaborative partner. Specifically, interacting with a member Y who had higher (or lower) discount rates was associated with member X's post-collaborative discount rates being higher (or lower), adjusting for their pre-collaborative discount rates. also includes other evidence of direct social influence. These influences include the multiplicative pathway from member X's pre-collaborative preferences to member Y's post-collaborative preferences (B = 0.07 [0.018, 0.161]) and member Y's pre-collaborative preferences to member Z's post-collaborative preferences (B = 0.13 [0.033, 0.318]). The pathway from member Z's pre-collaborative preferences to member Y's post-collaborative preferences did not quite reach statistical significance based on the bias-corrected confidence interval including zero (B = 0.13 [− 0.015, 0.388]). Though these above effects derive from joint, multiplicative pathways in the model, they are all still considered direct social effects because they trace the social influence between network members who interacted directly at some point during the chain sequence. Direct and indirect social influence derived from the path model in CL confidence limit. Confidence limits derived from 95% bias-corrected bootstrap confidience intervals based on 10,000 resamples. The significance of the path coefficients ( a ) was based on whether the confidence interval included zero or not ## Dependent variable ## Path Path labeling (see ## Indirect social influence The indirect social effect of interest was the influence of network member X on member Z, due to them never directly interacting during the study. The joint, multiplicative pathway between member X's pre-collaborative discount rates and member Z's post-collaborative discount rates was significant (B = 0.07 [0.021, 0.187]). That is, network member X's preferences uniquely predicted Dyad XY's decision making, which uniquely predicted Dyad YZ's decision making, which then uniquely predicted member Z's post-collaborative decision making. Similar to above, because autoregressive paths were included in the model (i.e., Ind Z Pre → Ind Z Post), these social influence effects on network member Z's postcollaborative decision preferences are after adjusting for member Z's baseline preferences. To further test the indirect influence of network member X on network member Z, a reduced path model was fit to the data (see . In this model, a path was included between member X's pre-collaborative discount rates (Ind X Pre) and Dyad YZ's discount rates, and a path between Dyad YZ and member Z's post-collaborative discount rates (Ind Z Post). The significant path between Ind X Pre and Dyad YZ (B = 0.23 [0.034, 0.419]) more clearly demonstrates the social contagion of decision preferences due to member X not being directly involved in the Dyad YZ collaboration phase. The joint multiplicative effect of these two pathways was also significant (B = 0.19 [0.058, 0.432]), again indicating a significant indirect social influence of network member X on network member Z. # Discussion The current study utilized small social networks to measure both direct and indirect social influence on temporal discounting in a controlled, laboratory setting. Participants' post-collaborative discount rates were predicted by direct social interaction with another dyad member, supporting previous research [bib_ref] Age-related differences in delay discounting: Immediate reward, reward magnitude, and social influence, Bixter [/bib_ref] [bib_ref] Are intertemporal preferences contagious? Evidence from collaborative decision making, Bixter [/bib_ref]. These results mean that collaborating with a high temporal discounter was associated with a network member exhibiting higher discount rates post-collaboratively, even adjusting for the network member's baseline discount rates. The additional, novel finding of the current study is the presence of indirect social influence on temporal discounting. This was exhibited by a significant pathway connecting the decision preferences of two network members that did not share a direct link or interaction with one another. These results demonstrate that temporal discounting can be socially transmitted through intervening network members (i.e., social contagion). How can decision preferences, such as temporal discounting, propagate throughout a social network? One explanation derives from prior research that showed a convergence effect in group temporal decision making, in which participants' preferences are more similar following direct social interaction [bib_ref] Age-related differences in delay discounting: Immediate reward, reward magnitude, and social influence, Bixter [/bib_ref] [bib_ref] Are intertemporal preferences contagious? Evidence from collaborative decision making, Bixter [/bib_ref]. This effect suggests that participants revise their preferences to be partially aligned with the preferences of their collaborative partner. As a result, when an individual then goes on to collaborate with a new network member, they are not solely bringing their own baseline preferences to the collaboration, but instead bring a mix of their own preferences and the preferences of the original network member. In the context of the current study design (see , after member Y collaborates with member X, member Y enters the second dyad collaboration (Dyad YZ) with preferences that are revised to be partially aligned with member X's preferences. Insofar as member Z revises their own preferences based on the interaction with member Y, there still exists a trace of member X's preferences in the behavior that is observed during the Dyad YZ collaboration. The results of the reduced path model shown in most clearly demonstrate this idea. Member X's baseline discount rates predicted Dyad YZ's discount rates, even though member X is not directly involved in that latter dyad. Presumably, network member Y's behavioral influence on Dyad YZ included the previous influence of collaborating with member X. Prior research on the social contagion of behavior often utilized observational study methods (e.g., [bib_ref] Exercise contagion in a global social network, Aral [/bib_ref]. Studies that deal with pre-existing The reduced path model that measured the indirect social influence of network member X on network member Z through their shared link with member Y. Member X's pre-collaborative discount rates (Ind X Pre) significantly predicted Dyad YZ's discount rates even though member X was not directly involved in that collaboration. The significance of the path coefficients (*) was based on whether the 95% bias-corrected bootstrap confidience intervals included zero or not social networks in uncontrolled environments have difficulty controlling for homophily and context effects. Both of these latter phenomena can provide alternative explanations for social contagion effects [bib_ref] Is obesity contagious? Social networks vs. environmental factors in the obesity epidemic, Cohen-Cole [/bib_ref] [bib_ref] Homophily and contagion as explanations for weight similarities among adolescent friends, De La Haye [/bib_ref]. Studying structured social networks in laboratory environments help to isolate the presence of social influence effects. In these controlled environments, indirect social influence is sometimes not observed in decision-making behavior (e.g., [bib_ref] Cooperation and contagion in web-based, networked public goods experiments, Suri [/bib_ref]. In the present study, focusing on decisions about delayed rewards, indirect social influence was observed and could be better attributed to social contagion. This is because control was placed on the order of the social interactions to ensure paths of influence could only flow in one direction. Specifically, network members X and Y first interacted with one another, followed by members Y and Z. Effort was made to ensure that there was no direct interaction between members X and Z. Therefore, any indirect effect between X and Z's decision preferences can be better attributed to the transmission of preferences through member Y. Future research will need to test the size, scope, and generalizability of the social contagion of decision making. For instance, observational network studies have observed social influence up to three degrees of separation [bib_ref] The spread of obesity in a large social network over 32 years, Christakis [/bib_ref] [bib_ref] Social network determinants of depression, Rosenquist [/bib_ref]. Laboratory studies can extend the current findings to measure the distance that indirect social influence can spread through small social networks in controlled environments. Moreover, it will be necessary for future research to study social network effects on decisions that more accurately model the everyday decisions that people encounter. For instance, the task used in the current study dealt with hypothetical monetary rewards. Though previous studies have often found no significant differences between individual discounting tasks that used real vs. hypothetical rewards [bib_ref] Within-subject comparison of real and hypothetical money rewards in delay discounting, Johnson [/bib_ref] [bib_ref] Delay discounting of real and hypothetical rewards, Madden [/bib_ref] [bib_ref] Statistical equivalence and test-retest reliability of delay and probability discounting using real..., Matusiewicz [/bib_ref] , and some group temporal decision-making studies have used financially incentivized tasks [bib_ref] How are individual time preferences aggregated in groups? A laboratory experiment on..., Tsuruta [/bib_ref] , future social network studies need to incorporate decisions that involve real consequences. These results will help to establish the practical impact of direct and indirect social influence on decision making and related behaviors. In addition to incentivized tasks, it will be helpful to study social network effects on more realistic decisions that involve a tradeoff between immediate and delayed gratification. Instead of repeated trials dealing with two arbitrary monetary rewards, tasks can involve decisions in a simulated market environment that involve consumption and savings. How consumer preferences spread among direct and indirect network links will provide a more complete picture of peer influences on everyday decision making. The overarching goal of the current study was to investigate the presence of social network effects on temporal decision making in a structured environment that controls for other extraneous factors. Now that social influence (both direct and indirect) has been shown to shape temporal decision preferences, it will be important for future research to focus on what variables impact the magnitude of this influence. For example, do different network structures produce more social influence and greater changes in temporal discounting? Does the degree of relationship between network members (e.g., close friends vs. strangers) moderate the magnitude of social influence? Once the focus is on assessing the magnitude of the change in temporal discounting, it will be helpful to have a nosocial interaction control group as a baseline comparison. Any observed changes in temporal discounting can then be contrasted with any naturalistic changes that may occur in decision preferences. The present study did not include a control group because it was an initial investigation into whether indirect social influence can be observed at all in small social networks. Now that social network effects are established in the temporal decision-making domain, the next step will be to test what structures or manipulations amplify this social influence compared to a baseline. Another area of future research is to see whether positive vs. negative behaviors propagate throughout social networks at different rates or magnitudes. Both positive behaviors (generosity; Fowler and Christakis 2010) and negative behaviors (e.g., drug use in adolescents; [bib_ref] The social contagion effect of marijuana use among adolescents, Ali [/bib_ref] demonstrate social network effects. Yet, direct comparison of the spread of behaviors based on valence is limited (cf., Dimant 2019). The judgment and decision-making literature is well suited for this and related research questions, however. Experimental tasks can manipulate the valence of the decision domain, such as decisions regarding gains versus losses, and measure whether some decision preferences are more socially contagious than others. Findings of valence effects will be relevant for intervention researchers and public health practitioners, depending on whether the goal is to facilitate the spread of socially desirable behaviors or limiting the spread of maladaptive behaviors. Due to individual differences in temporal discounting correlating with many consequential behaviors (e.g., addiction, obesity), recent studies have focused on measuring the impact of interventions on reducing high rates of discounting [bib_ref] Remember the future: Working memory training decreases delay discounting among stimulant addicts, Bickel [/bib_ref] [bib_ref] Change in delay discounting and substance reward value following a brief alcohol..., Dennhardt [/bib_ref]. Taking into account social network effects, some members may occupy more influential positions or nodes within local networks, making them desirable targets for behavioral intervention. In the laboratory, a recent study demonstrated the to which extent leaders and higher-status group members exert a stronger direct influence on others' decision preferences [bib_ref] Delay discounting in dyads and small groups: Group leadership, status information, and..., Bixter [/bib_ref]. Future research should take into account network characteristics and dynamics to leverage social influence and optimize intervention effectiveness. ## Supplementary information Supplementary information accompanies this paper at https:// doi. org/ 10. 1186/ s41235-020-00249-y. Additional file 1: Supplementary material. AcknowledgementsNone.Authors' contributionsMTB and CCK: development of theoretical background and research questions. MTB and CCL: designing the study. MTB: collecting data. MTB: performing statistical analyses. MTB and CCL: writing the manuscript. All authors read and approved the final manuscript.FundingThis work was partially supported by National Science Foundation Grant BCS1456928.Availability of data and materialsAll data and study materials are included as supplementary materials. These materials include the data (in .csv and .sav formats), the implementation of the path models using two software programs (AMOS, R), and the experimental task code (as a PsychoPy .py script).Ethics approval and consent to participateThis study was approved by the Institutional Review Board at Stony Brook University. All participants provided written consent prior to participating in the study.Consent for publicationNot applicable.Competing interests

Protocol for a Delphi consensus exercise to identify a core set of criteria for selecting health related outcome measures (HROM) to be used in primary health care Background: Promoting the collection and use of health related outcome measures (HROM) in daily practice has long been a goal for improving and assessing the effectiveness of care provided to patients. However, there has been a lack of consensus on what criteria to use to select outcomes or instruments, particularly in the context of primary health care settings where patients present with multiple concurrent health conditions and interventions are whole-health and person-focused. The purpose of this proposed study is to undertake a formal consensus exercise to establish criteria for selecting HROM (including patient-reported (PRO or PROM), observer-reported (ObsR)), clinician-reported (ClinRO) and performance related outcomes (PerfO) for use in shared decision-making, or in assessing, screening or monitoring health status in primary health care settings. Methods: A Delphi consensus online survey will be developed. Criteria for the Delphi panel participants to consider were selected from a targeted literature search. These initial criteria (n = 35) were grouped into four categories within which items will be presented in the Delphi survey, with the option to suggest additional items. Panel members invited to participate will include primary health care practitioners and administrators, policy-makers, researchers, and experts in HROM development; patients will be excluded. Standard Delphi methodology will be employed with an expectation of at least 3 rounds to achieve consensus (75% agreement). As the final list of criteria for selecting HROM emerges, panel members will be asked to provide opinions about potential weighting of items. The Delphi survey was approved by the Ethics Committee in the Faculty of Health Sciences at McMaster University. Discussion: Previous literature establishing criteria for selecting HROM were developed with a focus on patient reported outcomes, psychological/ behavioural outcomes or outcomes for minimum core outcome sets in clinical trials. Although helpful, these criteria may not be applicable and feasible for application in a primary health care context where patients with multi-morbidity and complex interventions are typical and the constraints of providing health services differ from those in research studies. The findings from this Delphi consensus study will address a gap for establishing consensus on criteria for selecting HROM for use across primary health care settings. # Background Collection of outcomes for use in evaluation of health care, quality improvement, clinical decision-making or effectiveness research continues to be promoted and encouraged. Routine collection of health related outcomes can occur in more formal contexts, for example in developing patient or disease registries, or less formal where findings are documented in individual patient charts. Collection of health related outcomes can be used at a system level or for individual patient management. In either case, the dilemma remains as to which health related outcomes to select that accurately captures the intended purposes for collection. There are a large number of criteria that could be considered to select outcomes which makes the process challenging. Several attempts to establish a minimum or core set of criteria used to evaluate or select outcomes have been reported [bib_ref] ISOQOL recommends minimum standards for patient-reported outcome measures used in patient-centered outcomes..., Reeve [/bib_ref] [bib_ref] Harmonized patient-reported data elements in the electronic health record: supporting meaningful use..., Estabrooks [/bib_ref] [bib_ref] Advancing the application, quality and harmonization of implementation science measures, Rabin [/bib_ref] [bib_ref] How to select outcome measurement instruments for outcomes included in a "Core..., Prinsen [/bib_ref] [bib_ref] Measuring vital signs: an IOM report on core metrics for health and..., Blumenthal [/bib_ref]. However, there are important limitations in these previous recommendations. Some focus predominately on patient reported [bib_ref] ISOQOL recommends minimum standards for patient-reported outcome measures used in patient-centered outcomes..., Reeve [/bib_ref] or psychological/ behavioural outcomes [bib_ref] Harmonized patient-reported data elements in the electronic health record: supporting meaningful use..., Estabrooks [/bib_ref] [bib_ref] Advancing the application, quality and harmonization of implementation science measures, Rabin [/bib_ref] ; there are many other types of measures used to assess the impact of care. Another attempt was developed in the context of comparing clinical trials and using an agreed upon set of common outcomes [bib_ref] How to select outcome measurement instruments for outcomes included in a "Core..., Prinsen [/bib_ref]. Similarly another is to be considered broadly at a systems level and not readily operationalized with consistency [bib_ref] Measuring vital signs: an IOM report on core metrics for health and..., Blumenthal [/bib_ref]. Although helpful, these previous criteria recommended to select outcomes may not be applicable for all types of outcomes or in other contexts in which they were developed. This paper will detail a protocol to establish criteria for selecting health related outcome measures (HROM) in the context of primary health care (PHC). PHC is characterized by patients with multifactorial health problems and accordingly the interventions are multilayered and complex. We propose a Delphi consensus study that includes experts in HROM development and clinical research. We provide background information around definitions and use of HROM, previous attempts to establish criteria to select measures, hypothesized benefits and use of HROM in clinical settings, the relevance to the PHC context and evaluation of complex interventions generally. The proposed research protocol details are specified and previous gaps in the literature are addressed. The overall goal of this research is to contribute to the development of a transparent and reproducible process to select HROM that will be used across different sites that are implementing and adapting complex PHC interventions. ## Purpose and definition of outcomes and health related outcome measures Apart from the practical and logistical aspects of routine collection of health related outcomes for evaluation purposes, there are some challenges conceptually in defining what is and is not an outcome. This is related to the variation in definitions of an outcome generally, as well as, definitions of an outcome measure and an outcome assessment [bib_ref] Guidance for industry: patient-reported outcome measures: use in medical product development to..., Guidance [/bib_ref] [bib_ref] The quality of care. How can it be assessed?, Donabedian [/bib_ref] [bib_ref] Systematic review of approaches to using patient experience data for quality improvement..., Gleeson [/bib_ref] [bib_ref] Primer on outcomes research, In [/bib_ref] [bib_ref] Patient-reported outcomes in cancer: a review of recent research and policy initiatives, Lipscomb [/bib_ref] [bib_ref] Patient-reported outcomes assessment in cancer trials: taking stock, moving forward, Lipscomb [/bib_ref] [bib_ref] Interpreting patientreported outcome results: US FDA guidance and emerging methods, Mcleod [/bib_ref] [bib_ref] Core outcome measures in effectiveness trials (COMET) initiative: protocol for an international..., Prinsen [/bib_ref] [bib_ref] Primary care: an increasingly important contributor to effectiveness, equity, and efficiency of..., Starfield [/bib_ref] [bib_ref] Contribution of primary care to health systems and health, Starfield [/bib_ref] [bib_ref] Factual errors about ClinicalTrials.gov and other federal mandates in special report by..., Zarin [/bib_ref]. The term outcome is often used in a global sense to connect the expected impact of an intervention. It is also used frequently to refer to instruments or scales used to assess or evaluate interventions or patient status at any point in the health care trajectory. The term outcome may be confused with the term endpoint (which sometimes is defined as a group of outcomes used to establish the benefit or harm of an intervention). For the purposes of this paper we have adopted the definition of an outcome as one that captures a "measureable characteristic that is influenced or affected by an individuals' baseline state or an intervention as in a clinical trial or other exposure" [bib_ref] Interpreting patientreported outcome results: US FDA guidance and emerging methods, Mcleod [/bib_ref]. An outcome could reflect both the benefit or harm to a patient who receives an intervention and exposure. This definition is in keeping with the Donabedian framework for quality improvement which defines outcome as the "effects of care on the health status of patients and populations" [bib_ref] The quality of care. How can it be assessed?, Donabedian [/bib_ref]. Outcome assessment is considered to be the most important of three categories of assessment of quality of care (including structure (context where care is delivered and including buildings, equipment, staffing, etc.) and process (interactions between patients, providers, system) [bib_ref] The quality of care. How can it be assessed?, Donabedian [/bib_ref] [bib_ref] Evaluating the quality of medical care, Donabedian [/bib_ref] [bib_ref] The quality of medical care: a concept in search of a definition, Donabedian [/bib_ref]. In this quality improvement framework, the focus of outcomes is on the impact of care on the person receiving it. Ideally the assessment of outcome of care should always reflect what patients actually experience and consider important. Thus HROM are measures or scales or instruments that are designed to capture outcomes (domains and constructs) in a health care context. What is key in this definition of outcomes and HROM is that it is not the specific type of measure or instrument or test that is used in the assessment but rather the intended purpose of the outcome measurement and the timing of the collection during care receiving. For example, consider hemoglobin A1c in the care of patients with diabetes. Capture of the frequency of measurement of hemoglobin A1c can be used to assess health service processes; estimation of the mean level (or mean change) can be used to assess the impact of the health services outcome (on the recipient). The influential Food and Drug Administration (FDA) in the United States considers a clinical outcome assessment as one that may use any of four primary types of "outcome measures" that would reflect domains important to relevant stakeholders and these include: patient reported outcome measures (PROM) or (PRO), observer reported outcomes (ObsRO), clinician reported outcomes (ClinRO), and performance outcomes (PerfO). In the FDA regulatory process, laboratory tests, biomarkers, and even mortality/survival are also important types of outcomes that can be used to assess the effectiveness of interventions (in their regulatory capacity, this refers to drugs, devices and biomarkers) for which approval is sought. This broad understanding of HROM and their potential classification are useful in understanding outcome assessment generally. ## Use of hrom as an intervention Promoting the capture of HROM in daily practice has long been a goal for assessing the effectiveness of care provided to patients. However, it is only recently that there is increasing recognition that the routine collection and interpretation of HROM in practice can be shown to influence ongoing care, such that it becomes an intervention in and of itself. Greenhalgh [bib_ref] The applications of PROs in clinical practice: what are they, do they..., Greenhalgh [/bib_ref] [bib_ref] The use of patient reported outcome measures in routine clinical practice: lack..., Greenhalgh [/bib_ref] hypothesized pathways of influence and impact of using PROM in clinical settings. Within this hypothesized model, the use of PROM (a type of HROM) will prompt clinicians to discuss health related quality of life concerns, it will enable clinicians to detect unrecognised problems, and will likely change the way in which a clinician will respond (as the findings from the PROM may show a change in the health related quality of life . The act of reviewing or using PROM serves to monitor treatment potentially resulting in changes to the patient's behaviour and improvement in the patient's overall health status or satisfaction with care. Thus, outcome assessment by collecting HROM not only evaluates the impact of an intervention at the resolution or end of a particular episode of care, but it can be considered an added "intervention" that serves to direct or modify care. For example, the rate of screening for depression is one outcome that could be used to demonstrate the impact of a routine visit to a family doctor. However, if the screening result is positive, then the physician will now direct care to address this previously undetected health problem. In the context of PHC, like other areas of health care provision, a wide spectrum of HROM are used to assess both simple and complex interventions and single and comorbid health conditions. Added to this complexity is that most visits to PHC are for symptoms or complaints rather than diseases so the range of available HROM related to single diseases are less appropriate [bib_ref] Effects of improved patient participation in primary care on healthrelated outcomes: a..., Sanders [/bib_ref]. It is not clear which of types of HROM should be selected to provide useful information to assess the impact of care or to allow clinicians and decision-makers to direct or modify care. What is an ideal HROM in one context may not be so in another context and in part reflects the intended purpose of the outcome assessment. The challenge for clinicians, researchers and decision-makers is to select the most appropriate HROM in light of the fact that likely several well established HROM currently exist and are candidates for selection. Although there have been some attempts to set some general guidelines for selecting HROM for outcome assessment, these criteria have several key limitations that have been noted and none provide recommendations for dealing with equally valid but competing measures. ## Hypothesized benefits of using hrom in clinical settings Donabedian was one of the first to propose that structure, process and outcomes are seminal to evaluating quality of care [bib_ref] Evaluating the quality of medical care, Donabedian [/bib_ref] [bib_ref] The quality of medical care: a concept in search of a definition, Donabedian [/bib_ref]. Since the late 1960s there has been great emphasis on the selection and use of HROM in quality assessment but also in effectiveness research and even directing clinical decision-making for individual patients. As noted previously, Greenhalgh [bib_ref] The applications of PROs in clinical practice: what are they, do they..., Greenhalgh [/bib_ref] [bib_ref] The use of patient reported outcome measures in routine clinical practice: lack..., Greenhalgh [/bib_ref] hypothesized pathways of influence and impact of using PROM in clinical settings. Within this model, PROM can be considered as having multiple purposes that include "clinical tests", "interventions" and "outcomes". [fig_ref] Table 1: Greenhalgh [24] model hypothesizing pathways of influence and impact in using PROM... [/fig_ref] details how the model specifies the clinical goal when using PROM and the potential impact of using these in clinical settings. Note that this model may be applied to HROM, as the definition of HROM includes PROM (as well as, ObsRO, ClinRO, PerfO). When considering the clinical goals (i.e., assess change, monitor, detect, etc.) for using an HROM, it is not clear if the criteria used to select measures will vary as a function of the specific purpose. It is likely that the criteria may vary not only as a function of the clinical purpose for using the HROM, but also the health domain being assessed by the tool and for other administrative concerns. Soliciting expert opinions on this issue would add to the knowledge of how to apply HROM for clinical, administrative, or research purposes. ## Primary health care context and complex interventions In the context of PHC, there is increasing recognition that most interventions are complex. The Medical Research Council (United Kingdom) has been instrumental in conceptualizing the issues related to developing, evaluating, and reporting on complex interventions. Complex interventions are characterized by having a number of interactions between components and requiring a number of different behaviours by those delivering or receiving the intervention. Adding to this definition is that a number of groups and organizational levels are affected by the intervention. Although some would make the distinction between the complexity of the intervention versus the complexity of the health system [bib_ref] Complex interventions or complex systems? Implications for health economic evaluation, Shiell [/bib_ref] the two components interact. Flexibility and adaptation characterize complex interventions. Practically this means that changes in implementation of an intervention are to be expected. More importantly, it follows that a number of outcomes may be required, as well as variability in outcomes assessing the same attribute of interest. This would suggest that adaptations of interventions in one component of a health system may result in the need to select different or modify existing HROM relative to that context. The benefit of this is that it reflects the varying and flexible aspects of PHC interventions and therefore reflects strengths of the interventions in different settings. The development of this Delphi consensus study is considered in the context of such a complex intervention. Health TAPESTRY is a novel intervention currently being implemented in Canadian PHC settings and undergoing evaluation in a randomized trial. The intervention components involve patients, volunteers, health care teams, and researchers, as well as, the use of an electronic patient health record accessed by all relevant stakeholders [bib_ref] A protocol for a pragmatic randomized controlled trial using the health teams..., Dolovich [/bib_ref]. There are several affiliates of Health TAPESTRY across different sites in Canada that vary in their patient populations and focus in PHC services. As expected, each Health TAPESTRY site currently uses different HROM (or will need to select new HROM), and this may present challenges for evaluating benefits and quality of this complex intervention. One overriding goal of Health TAPESTRY is to integrate new and old functionality within and across each of these centres. As the Health TAPESTRY intervention is being adapted and implemented it did not seem feasible to enforce standardization of HROM. However, stakeholders acknowledge that there is a need to guide selection of key HROM to be used in evaluating the impact of the Health TAPESTRY intervention across implementation sites. Health TAPESTRY serves as a quintessential example of PHC issues and the complexity of evaluation. Recognizing the potential for differences across Health TAPESTRY sites, a valid and trustworthy process to review and accept suggestions for HROM is needed to allow for evaluation of effectiveness and impact across sites. To our knowledge there is no guideline or consensus on criteria to select HROM within a PHC setting. ## Study purposes Our primary purpose for the Delphi consensus exercise is to derive a minimum core criteria set (CCS) of items to be used to select HROM when faced with many different measures that can be used within PHC settings. Related to this we will solicit opinions about the relative weighting of criteria groups to be used when selecting HROM. Additional purposes for the Delphi survey are to consider core areas and domains of HROM that are important to collect in PHC and reflecting the complex interventions that are flexible and adaptable (such as Health TAPESTRY). The CCS will assist in selecting HROM but it will not provide guidance as to the core health areas of importance for PHC. Therefore, a second purpose of this study is to elicit opinions about core areas and domains and HROM that effectively capture the attributes of interest within these core areas/domains that are optimal for use within PHC settings. # Methods/design ## General approach The stated objectives of this study require consensus on criteria for selecting HROM and opinions about areas and types of outcomes to be used in PHC settings and Health TAPESTRY. There are a number of methods used to solicit group opinion and consensus and these include nominal techniques, Delphi consensus, and consensus development conferences [bib_ref] Consensus development methods, and their use in clinical guideline development, Murphy [/bib_ref]. From these approaches we selected the Delphi consensus as the most appropriate for the purposes of this study. The global aim of the Delphi consensus exercise is to achieve consensus on a CCS for selecting HROM relevant to PHC and Health TAPESTRY. [fig_ref] Figure 1: Anticipated rounds of the Delphi consensus exercise and timing of soliciting opinions... [/fig_ref] shows the expected rounds of the survey to achieve consensus for CCS. Health TAPESTRY centres will also need to consider what key health areas or domains should be captured (for example physical function is a core area of health and within this there are several domains that might include activities of daily living and physical activity levels). We will solicit the opinions of the Delphi panel about core areas and domains and important HROM that they feel meet the CCS and are relevant for PHC settings and complex interventions such as Health TAP-ESTRY. We will not require consensus with regards to core areas and domains suggested by participants. ## Key assumptions and definitions defining hrom The types of HROM we are considering in this Delphi consensus study are likely standardized measures of health status, disability, impairment, or handicap, as well as other clinical tests. They assess specific dimensions of a disease or a health problem (e.g., depression or frailty or fasting glucose levels). Many of these measures can be uni-dimensional (reflecting a single health construct) or multi-dimensional (several health constructs). A number of different measures reflecting the patients' health experiences can be used in the PHC setting and these include: ## Defining primary health care We have selected the PHC care context for our study, as it encompasses a general approach to health policy and service provision that reflect the core principles espoused by the World Health Organization (universal access, health equity, community participation, inter-sectoral approaches to health) [bib_ref] Primary care (PC) and primary health care (PHC), Muldoon [/bib_ref] and recognizes the importance of the broad determinants of health. PHC focuses on patient and provider relationship, as well as organized care. Thus, PHC embraces a wide suite of services and involves a broad range of health care providers [bib_ref] Can we decide which outcomes should be measured in every clinical trial?..., Idzerda [/bib_ref] that include the following: i) family physicians/general practitioners; ii) nurses, nurse practitioners; iii) rehabilitation professionals (e.g., occupational therapist); iv) physician assistants; v) nutritionists and; vi) behaviour counselors (e.g., social work). The selection of HROM in the context of PHC can encompass a variety of outcomes reflecting domains of efficacy, effectiveness, patient engagement, patient satisfaction, and other aspects of care. Given the range of health care providers, the intended purposes of the use of the outcome may also vary. Further the selection of outcomes may need to reflect the series of complex interactions and interventions that are part of the services provided in PHC. Selecting core areas and domains from which to map outcomes Core areas and domains represent the key aspects of health that the HROM attempts to capture. For example, physical function is a core area of health. Domains within the core area of physical function could include physical activity or lower extremity mobility. It is anticipated that a community adapting an intervention or putting a new intervention in place may choose different HROM that capture important core areas and domains relevant to PHC. As noted previously, our literature review identified one initiative to develop criteria for selecting outcomes in the context of clinical trials and the need for developing a core outcome set (COS) [bib_ref] Can we decide which outcomes should be measured in every clinical trial?..., Idzerda [/bib_ref]. This review stemmed from the recent work from Core Outcome Measures in Effectiveness Trials (COMET) [bib_ref] The COMET initiative database: progress and activities from, Gargon [/bib_ref] [bib_ref] Developing core outcome sets for clinical trials: issues to consider, Williamson [/bib_ref] and OMERACT [bib_ref] The COMET initiative database: progress and activities from, Gargon [/bib_ref] [bib_ref] Toward a generalized framework of core measurement areas in clinical trials: a..., Boers [/bib_ref] [bib_ref] Developing core outcome measurement sets for clinical trials: OMERACT filter 2.0, Boers [/bib_ref] [bib_ref] Updating the OMERACT filter: core areas as a basis for defining core..., Kirwan [/bib_ref] suggesting approaches to developing core areas and domains for selecting outcomes. The aim of this approach is to establish key areas of health and then select HROM to consistently assess across different research and clinical initiatives. The challenge with selecting a core set of HROM is that most initiatives are centred on a single disease and in the PHC context there are multiple health conditions often for the same patient. We have specified a broad set of core areas (see [fig_ref] Figure 2: Core areas for health-related outcome measures [/fig_ref]. The literature suggests that the selection of HROM is related to the purpose and core area/domain of health that is of interest. At this time there is no literature specifying core areas/domain of relevance to PHC. Therefore a secondary aim of the Delphi consensus survey is to solicit opinions of the Delphi panel on the relative value of the core areas, domains and subdomains that has been preselected by Health TAPESTRY investigators. Additionally, we will solicit opinions on important HROM that would capture the aspects of health of interest and meet the core set of selection criteria established through the Delphi consensus exercise. Opinions about core areas, domains and subdomains, as well as suggested HROM will assist different Health TAPESTRY centres in selecting these measures (once the core criteria are established). ## Development of the delphi questionnaire for ccs ## Literature review The investigators assembled potentially relevant criteria for selection of outcomes from a targeted literature review. We searched Pubmed using terms related to outcomes (i.e., outcome measures, Patient reported outcomes, Comparative effectiveness, Patient-centered outcomes research, Psychometrics, Measurement properties, Questionnaire, Patient Registry, Methods). We also searched specific websites (i.e. FDA and others listed below) related to outcome measures. The citations listed below were important sources of from which our criteria was selected and these included: ## 1) consensus-based standards for the selection of health Measurement Instruments (COSMIN) checklist) [bib_ref] Inter-rater agreement and reliability of the COSMIN (COnsensus-based standards for the selection..., Mokkink [/bib_ref] [bib_ref] The COSMIN checklist for evaluating the methodological quality of studies on measurement..., Mokkink [/bib_ref] [bib_ref] The COSMIN study reached international consensus on taxonomy, terminology, and definitions of..., Mokkink [/bib_ref] [bib_ref] The COSMIN checklist for assessing the methodological quality of studies on measurement..., Mokkink [/bib_ref] [bib_ref] Rating the methodological quality in systematic reviews of studies on measurement properties:..., Terwee [/bib_ref] ; the checklist establishes which measurement properties should be evaluated in health related patient-reported outcomes and is used to evaluate studies that attempt to establish the properties of a health related patient-reported outcomes.. ## Structure of the consensus survey The criteria derived from the targeted literature search (detailed above) were grouped into four primary categories reflecting aspects of the outcome and these included: 1) gold standard measurement properties; 2) purpose and structure; 3) applicability; and 4) feasibility. shows some examples of subcategories of criteria within the four broad categories. shows the criteria proposed to Delphi panel members for their consideration. From the targeted literature search a total of 35 criteria have been identified for Panel members to consider in Round 1 (see . In Round 1 panel members are asked to rate whether they view the criterion as one that could be used to select amongst different HROM and then asked to comment on their reasons. For each criterion, Delphi participants can view a short description of the conceptual basis/definitions and where possible references or sources associated with it were noted. For criteria that has reached consensus, Delphi participants are asked additional questions in Round 2. Delphi participants are asked to comment on potential for differences in inclusion or exclusion of criterion relative to the four clinical goals/scenarios (i.e., engage, assess, screen, monitor). Prior to launch of the Delphi consensus survey pre-testing will be undertaken to ensure correct functioning of the survey online screens and for wording of the questions and notes provided to participants. General approach for soliciting opinions about core areas in round 3 Developing questions related to core areas and domains of outcomes shows core areas and domains that the investigators selected after review of the literature of frameworks for core outcome measures [bib_ref] Can we decide which outcomes should be measured in every clinical trial?..., Idzerda [/bib_ref]. The investigators believe these core areas are comprehensive and important to PHC. We will solicit opinions from the Delphi panel regarding suitable domains and subdomains to be captured consistently in primary care. We will also solicit opinions about HROM suitable for primary care settings within the suggested domains. General structure of questions about the weighting of core items These questions will be open ended and will be determined based on the results of the previous 2 rounds. ## Selection of stakeholder groups and nominated participants Our general approach is to select individuals who have knowledge of the outcome and outcome assessment and with clinical or research experience in PHC [bib_ref] The Delphi technique: Past, present, and future prospects, Hasson [/bib_ref] ; the goal is also to have representation of different perspectives with respect to the use of HROM or the professional role of those who use the HROM [bib_ref] The Delphi technique: Past, present, and future prospects, Hasson [/bib_ref]. For this reason we will use a purposive sampling approach. The selection of Delphi panel members should reflect the population that is intended to use the CCS or be informed by the research. In order to ensure validity of the final CCS, the panelists should reflect a diverse range of stakeholders, and likely from representing different geographic regions within Canada or internationally. ## Identifying relevant stakeholders There are two sets of issues to consider when selecting participants in the Delphi exercise to achieve consensus for the selection of criteria for choosing outcome measures in PHC settings and Health TAPESTRY. The first is with regards to the stakeholders and potential areas of expertise or representation from which to recruit potential panel members. We will identify key stakeholder Four clinical purposes of using outcomes and these include activities that Engage, Assess, Screen, and Monitor primary health care patients groups and these include: i) PHC team members (all health professional disciplines of relevance) including those likely to be involved in the Health TAPESTRY across Canada (including sites in Ontario, Quebec, Saskatchewan, NFLD, and British Columbia); ii) management or administration team members in PHC clinical care sites; iii) health and health services researchers with experience in primary care HROM (including quality indicators); iv) experts in administrative issues related to collecting measures (including harmonization related issues); v) methodologists working in outcomes-related and questionnaire development research; and vi) community service organizations involved in patient care or support. Patients and volunteers will not be represented in the Delphi panel membership. ## Invitation and reminder to delphi panel participants All Delphi participants identified will be invited to complete each Delphi round, unless they indicate withdrawal from the study. Continuation of the Delphi participant (by linking to the survey) will be assumed as consent to participate. Reminders will be sent every 14 days following distribution of the survey. Round 1 will be closed after 12 weeks. ## Sample size for the delphi panel There is no set standard for sample size of a panel but it is generally agreed that the more members will increase the reliability of group judgments [bib_ref] Consensus development methods, and their use in clinical guideline development, Murphy [/bib_ref]. In has been suggested that a minimum number of panel members [bib_ref] The Delphi method as a research tool: an example, design considerations and..., Okoli [/bib_ref] would range from 10 to 18 panel members per area of expertise. Given the complexity of the criteria and the number of different health professionals likely with multiple areas of expertise, we will aim towards a minimum sample size of 50 participants. We will send email invitations to approximately 60 potential panel members assuming a 20% rejection rate, yielding a final sample size of 50 panel members. We are also allow for a "snowball sampling" approach, which we anticipate will increase our sample; we will encourage the pool of potential panel members to send invitations to other potential relevant participants. ## Sampling strategy and response rate A combined strategy will be used to select Delphi panel members. A purposive sample of panel members representing relevant professional and administrative stakeholders within PHC will be selected. A snowball sampling approach will also be used to recruit potential panel members for the Delphi consensus exercise. We will ask the purposive sample of panelists to suggest names (and email contact) of other relevant stakeholders. Potential panel members who have experience in PHC practice or outcome measurement research will be considered. A response of 75% is suggested for each round to achieve or maintain rigour [bib_ref] Research guidelines for the Delphi survey technique, Hasson [/bib_ref] and we will monitor response rate for each round. There is the potential to introduce "withdrawal bias" if participants choose to withdraw after the first round. If Delphi panel members choose to not continue participation, we will attempt to solicit reasons for withdrawal (e.g., not enough time, too complicated, not interested) in order to assess possible threats to validity of the Delphi process [bib_ref] Consulting the oracle: ten lessons from using the Delphi technique in nursing..., Keeney [/bib_ref]. ## Invitation, reminders and data management of panel member information Each participant will be allocated a random identification number and analysis of results through LimeSurvey 2.0. Demographic data (collected at the end of Round 1) will be collated and summarized as exported by Lime-Survey 2.0. ## Selection role of the steering committee members Steering committee members will be selected from our team of investigators. The Steering Committee will function to summarize responses from the iterative Delphi consensus rounds. The Steering Committee will be responsible for preparing group feedback to panel respondents, and to identify any concerns with moving forward to reaching consensus. Although the intent of the Delphi consensus exercise is to allow panel members to judge and filter the information provided, the Steering Committee may need to make some decisions to reduce items if consensus is not achieved on many criteria. This approach may be necessary to prevent risk of over burden to Delphi panelists for subsequent rounds [bib_ref] Research guidelines for the Delphi survey technique, Hasson [/bib_ref]. A reduction in items will be communicated to panel members and the opportunity to respond to the feedback will be provided. This will ensure that any potential for bias is identified and rectified in subsequent rounds [bib_ref] Research guidelines for the Delphi survey technique, Hasson [/bib_ref]. # Delphi consensus methods The Delphi method has been used in a variety of research contexts to achieve both consensus [bib_ref] Consensus development methods, and their use in clinical guideline development, Murphy [/bib_ref] and dissensus [bib_ref] The Delphi method as early warning: linking global societal trends to future..., Van De Linde [/bib_ref] when soliciting stakeholder or expert opinion. The Delphi approach, is typically selected as an unbiased process to solicit opinions and reach consensus when there is no clear evidence for the solution to the problem under consideration. Stakeholder or expert (commonly referred to as the panellists, participants or respondents) consultation is required. Developed in the 1950's and 60's in a RAND study [bib_ref] Research guidelines for the Delphi survey technique, Hasson [/bib_ref] the methodology has been key aspects that include: i) anonymity of panel members; ii) feedback is controlled and fed back to panelists; iii) survey and feedback is iterative (usually 3 successive rounds); and iv) iteration of rounds until consensus is achieved (or the law of diminishing returns sets in). The advantages of the Delphi process are that opinions of experts and other stakeholders are collected in a non-confrontational manner, free from group pressures, while group opinions are fed back to panelists. The process may help to identify aspects of the problem or solution under consideration that may have been missed or may have thought to be of limited relevance. ## Defining and achieving final consensus and timeline For the purpose of this study, consensus is defined as general agreement of a substantial majority (75% or greater) of Delphi panelists. Re-evaluation of items would continue with iterations of Delphi rounds (a minimum of 2) or until consensus is reached on all criteria within the four groupings. If consensus cannot reached after 5 rounds, the Steering Committee will stop rounds and finalize the items within the CCS. In accordance with improved reporting standards, consensus is thus defined a priori, with specific quantitative thresholds to achieve consensus, with rounds specified as an expected 3 and up to 5, criteria for excluding and including criteria and feedback on why items were dropped or added [bib_ref] Defining consensus: a systematic review recommends methodologic criteria for reporting of Delphi..., Diamond [/bib_ref]. We have specified how we will invite and select Delphi participants in a manner that is reproducible [bib_ref] Defining consensus: a systematic review recommends methodologic criteria for reporting of Delphi..., Diamond [/bib_ref]. It is anticipated that we will require three rounds and expect analysis between the rounds will take up to 12 months. Round 1 commenced on January 15 2016. ## First round As the Delphi method involves a series of rounds in order to achieve consensus, different activities will occur at each successive round. In the first round, panel members will link to a web-based version of the Delphi questionnaire in LimeSurvey 2.0, which includes background information, instructions to the survey, and help files for specific questions. In this Round 1 Delphi panelists will be asked to rate: the appropriateness of the criterion for selecting HROM, and 2) the reason why the item would be selected or not. We will ask Delphi panel members to suggest rephrasing, provide any rationale for their choices (each item in the survey has a comment box), and suggest missing or new items. Panel members will be given up to 12 weeks to respond. To enhance response rate, panelists will be sent a reminder notice after 2 weeks of the initial email requesting participation. ## Second round The responses from Round 1 will be aggregated, and analysed. The aggregated information will be fed back to panel members anonymously as part of the introductory material for the second round. Items where there is consensus to include in the CCS will be identified. Conversely, items for which there is consensus to exclude will also be identified. Finally, items for which there is a lack of consensus will be identified. Panel members will then be asked to reconsider the criteria for which consensus will not have been reached. Delphi panel members will be asked to review these findings and provide any comments. Additionally, in the second round respondents will be asked if priority ranking of the criterion will change as a consequence of the clinical goal (see [fig_ref] Figure 1: Anticipated rounds of the Delphi consensus exercise and timing of soliciting opinions... [/fig_ref]. identifies four clinical goals (adapted from Greenhalgh [bib_ref] The applications of PROs in clinical practice: what are they, do they..., Greenhalgh [/bib_ref] [bib_ref] The use of patient reported outcome measures in routine clinical practice: lack..., Greenhalgh [/bib_ref] that are related to the HROM being used. This will be asked for items for which there is consensus that the criteria should be included. Note that consensus is not necessary in these responses to priority ranking. Our aim is to solicit opinions with regards to ranking as a function of the clinical goal of the HROM. We will ask Delphi panel members to suggest rephrasing, provide any rationale for their choices (each item in the survey has a comment box), and suggest missing or new items. ## Third round (or greater) Criteria that remain indeterminate from Round 2 will be brought back into the third round or for continued series of iterations until consensus is achieved (see [fig_ref] Figure 1: Anticipated rounds of the Delphi consensus exercise and timing of soliciting opinions... [/fig_ref]. Panel members will re-evaluate criteria for which there is insufficient consensus for inclusion/exclusion. Conversely, a third round will be omitted if consensus (on all remaining items) is reached following the second round. We will ask Delphi panel members to suggest rephrasing, provide any rationale for their choices (each item in the survey had a comment box), and suggest missing or new items. We will add additional questions regarding the relative weighting of the four groupings of items for a final CCS for which consensus has been reached. These questions will be developed as the CCS emerges following the Delphi rounds. The aim of these questions is to solicit opinions only and not achieve consensus. Finally, we will solicit opinions about the core areas and domains and outcomes that meet the CCS final criteria within these domains. # Discussion There are several important gaps in the literature with respect to how to select criteria to choose HROM and in which contexts. We consider several of these gaps here. ## Previous attempts to establish criteria for selecting hrom and their limitations There have been several attempts to establish criteria for selecting HROM. If the focus of the outcome assessment is on matters of importance to patients, then it is clear that measures evaluating health related quality of life are important to consider. The ISOQOL has provided one such effort with the goal to establish minimum standards for the design and selection of PROM suitable for comparative effectiveness reviews and for patient centered outcomes research [bib_ref] ISOQOL recommends minimum standards for patient-reported outcome measures used in patient-centered outcomes..., Reeve [/bib_ref]. They assembled potential criteria from a targeted literature review identifying existing guidance (n = 28 guidance documents) on the selection of PROM in patient-centred outcomes research, followed by an online survey of their membership. Their recommended criteria focused on a succinct list (n = 7) of measurement properties (i.e., reliability, validity, etc.) and practical attributes (i.e., interpretability, acceptable burden, etc.). Although an important initial step, these criteria were developed with consideration of PROM and multidimensional instruments reflecting health related quality of life; they did not take into account the specific health setting or clinical context for use (i.e., screening, monitoring, etc.). A second attempt to establish criteria for selecting HROM did take into account the PHC context; the criteria for selecting outcomes was considered with respect to use of the electronic health record to record PROM specifically measuring health behaviours and psychosocial concerns [bib_ref] Harmonized patient-reported data elements in the electronic health record: supporting meaningful use..., Estabrooks [/bib_ref] [bib_ref] Advancing the application, quality and harmonization of implementation science measures, Rabin [/bib_ref]. In the inaugural phases of this initiative, content experts (i.e., expertise in physical activity, goal setting, patient literacy, anxiety and depression, etc.) were instructed to consider a set of proposed scientific and practical criteria for recommending outcomes to capture attributes of health promotion and disease prevention. From these a core set of criteria (n = 12) that were grouped according to "scientific" attributes and "practical considerations" were proposed. A grid-enabled measures (GEM) portal on a wiki site provided a repository of recommended psychosocial outcome instruments and allowed other stakeholders (researchers, practitioners, administrators) to continue to make suggestions and recommendations on the use of these measures in the context of these criteria. Although the focus of these criteria was targeting only selection of PROM for behavioural and psychosocial issues and considering the electronic health record in PHC, real world contexts were considered and reflected in the "practical considerations" components of the criteria. A third set of core criteria were developed by the Core Outcome Measurement Instrument Selection (COMIS) [bib_ref] How to select outcome measurement instruments for outcomes included in a "Core..., Prinsen [/bib_ref] [bib_ref] Core outcome measures in effectiveness trials (COMET) initiative: protocol for an international..., Prinsen [/bib_ref] in cooperation with the COMET Initiative and the COSMIN. The criteria was derived using a Delphi consensus exercise (that included both research and clinical experts) with the intended goal of developing a guideline on how to select measurement instruments included in a Core Outcome Set (COS). A COS is an agreed minimum set of outcomes that should be measured and reported in all clinical trials of a specific disease or trial population. The definition of an outcome considered in this research is one that conceptualizes outcome as the "what" is being measured and defined as a "construct or domain". It follows then that an outcome measurement instrument captures these constructs. [bib_ref] How to select outcome measurement instruments for outcomes included in a "Core..., Prinsen [/bib_ref] [bib_ref] How to select outcome measurement instruments for outcomes included in a "Core..., Prinsen [/bib_ref] did not restrict their criteria to any type of instrument (e.g. included PROM but is all inclusive) and included experts with both clinical and research experience. However, the focus during development of these criteria was to develop a minimum set of outcomes for use in clinical trials for a disease or patient population rather than a health service or setting. The fourth initiative to identify criteria to select health related measures generally and HROM was developed by the Institute of Medicine (IOM) which examined core measures used within the American health care system and considered some criteria for core measures and core sets. The IOM committee aimed to decrease inconsistent and duplicative efforts to collect HROM when assessing system level quality and established criteria for core measures [bib_ref] Measuring vital signs: an IOM report on core metrics for health and..., Blumenthal [/bib_ref]. For individual patient outcomes these included: consideration of the measures' importance to health, strength of linkage to progress, understandability of the measure, technical integrity, potential for broader system impact, and utility at multiple levels. Additional consideration was given to system related issues and these included: systemic reach, outcomes oriented, person meaningful, parsimonious, representative, and utility at multiple levels. The IOM effectively considered both individual and system level categories that could be applied to PHC and likely capturing complex interventions. These previous attempts to establish criteria to select PROM/HROM demonstrate the growing interest in establishing criteria and also justification for selecting outcomes. These previous attempts provide important context for what criteria have been considered and how these were developed. There are areas of overlap across these four different sets of recommended criteria which reflect a convergence of key attributes that may be grouped into those reflecting measurement properties and practical aspects. However, there are differences in items within these broad categories across criteria sets, which may in part reflect the specific purposes of each of their respective development processes. The influence of the health care setting, particularly PHC and the complex nature of this environment, is not clear in the development of these criteria and would merit further exploration. Although helpful, these previous attempts at establishing a CCS may not be applicable and feasible for application in PHC settings where patients with multiple health conditions and complex interventions are the typical and the constraints of providing health services differ from those in research studies. We have proposed a methodology to conduct a Delphi and constructed an online survey which we pilot tested for comprehension and pre-tested to ensure adequate functioning of the survey set up. Although we did not undertake cognitive debriefing but believe the pre-testing will take this into account. Understanding the context of primary care and primary health care There is some inconsistency in understanding differences between primary care and PHC [bib_ref] Primary care (PC) and primary health care (PHC), Muldoon [/bib_ref] and the differences are typically around the types of services provided. For the purposes of this paper we refer to primary care where there are interactions and services with clinicians (usually general practitioners) as first line of service. [bib_ref] Primary care (PC) and primary health care (PHC), Muldoon [/bib_ref]. In contrast PHC is broader in scope and encompasses a general approach to health policy and service provision (i.e. core principles that include universal access, health equity, community participation, inter-sectoral approaches to health promoted by the World Health Organization) [bib_ref] Primary care (PC) and primary health care (PHC), Muldoon [/bib_ref]. Effective PHC is community-based, promotes healthy lifestyles to prevent illnesses, considers ongoing care for chronic conditions and acknowledges the importance of the broad determinants of health. There is evidence to show that PHC helps prevent morbidity and mortality and is associated with more equitable distribution of health in the populations [bib_ref] Primary care: an increasingly important contributor to effectiveness, equity, and efficiency of..., Starfield [/bib_ref]. PHC also reflects the first contact with the health care system and coordination and integration thereby ensuring access to care (diverse services and health providers)that include the following: i) family physicians/ general practitioners; ii) nurses, nurse practitioners; iii) rehabilitation professionals (e.g., occupational therapist); iv) physician assistants; v) nutritionists and; vi) behaviour counselors (e.g., social work). Understanding the role of PHC in the health of a population, the rationale for selecting HROM is compelling. The selection of HROM in the context of PHC can encompass a variety of outcomes reflecting domains of efficacy, effectiveness, patient engagement, patient satisfaction, and other aspects of care. Given the range of health care providers, the intended purposes of the use of the outcome may also vary. Further the selection of outcomes may need to reflect the series of complex interactions and interventions that are part of the services provided in PHC. ## Availability of multiple hrom We anticipate that there are many HROM that possess good aspects and this presents a new dilemma; that is, selecting from several HROM attempting to capture the same health attribute of interest that meet the established criteria for selection. The stakeholder is faced with having a rationale for their choice. One potential strategy to address this issue is to consider "weighting" some items as more important over others; soliciting opinions from the Delphi panel on the final set of criteria for which consensus is achieved may provide some practical direction for implementation. Soliciting opinions about important core domains for outcomes may also assist stakeholders in making choices amongst different HROM that meet the CCS. The conceptual models that underpin some complex or multi-dimensional HROM may need to be compared to core areas deemed appropriate or ideal for the PHC context. As noted previously, primary care patients reflect heterogeneous population with a variety of health conditions. There is limited discussion in the literature about the core areas and subdomains that ideally should be captured. Opinions from panel members will be collated and reflected back in the guidance document of for implementation of the CCS. Because of these gaps, we developed this protocol to undertake a Delphi consensus online survey to establish criteria for a broader array of types of outcomes (i.e., HROM) specific to the context of PHC settings. A Delphi consensus exercise is a preferred method when consensus is lacking and in this case the literature suggests some lack of overlap in possible criteria. The advantages of the Delphi which include anonymity, provision of controlled feedback, and avoidance of face to face meetings will minimize potential biases due to personalities and other types of influences. Our selection of sample members will not be random and is influenced by current members of different Health TAP-ESTRY affiliates; the sample therefore may reflect these perspectives and not be truly representative of all primary health care contexts. The findings from this Delphi study will address a gap for establishing consensus on criteria for selecting HROM for use across PHC settings and complex interventions. # Funding Funding for this study is from Health Canada and the Canadian Institutes of Health. Thes agencies had no role in the design of this protocol and will not be involved in the future data collection, analysis and interpretation of data, nor in the writing of the manuscript. Authors' contributions PLS, LD, AG, and LL were responsible for the initial methods development and all authors contributed to refining these and comments on the results. All authors (PLS, LD, AG, LL, DO, LG, JR, DM, MK and PR were responsible for reviewing drafts of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Approval for this Delphi study was obtained from the Research Ethics Committee (RB 15-207) at McMaster University, Ontario, Canada. Delphi participants will be required to consent to participation in the online survey. [fig] Figure 1: Anticipated rounds of the Delphi consensus exercise and timing of soliciting opinions on clinical scenarios and core outcomes [/fig] [fig] Figure 2: Core areas for health-related outcome measures (HROM) to be used in primary health care [/fig] [fig] Figure 3, Figure 4: Categorization (grouping) of criteria to consider for selecting outcomes in primary health care; some examples of criteria within each categorization are shownTable 2Criteria found in targeted literature search and the items identified as potential criteria to be used when selecting [/fig] [table] Table 1: Greenhalgh [24] model hypothesizing pathways of influence and impact in using PROM in clinical settings [/table] [table] 1: PROM (e.g., visual analogue scale [VAS], Short Form 36 [SF36]): PROM's are measures that are reported or collected directly from the patient. PRO/PROM may be collected via self-administered questionnaires completed by the patient themselves or via interviews of the patients ensuring their views are adequately captured. 2. PerfO (e.g., Timed up and go test): PerfO are those that require assessment of the patient's capacity to perform pre-specified tasks. Typical performancebased measures include the Berg Balance Scale or the 50 ft Walking Test. 3. ClinRO/ ObsRO (e.g., Edmonton Frailty Index, Functional Index Measure [FIM]): Outcomes that include clinical scales are measures where clinicians/ outcome assessors evaluate the health construct of interest. This can include laboratory tests. [/table]

A seeded propagation of Cu, Zn-superoxide dismutase aggregates in amyotrophic lateral sclerosis # Introduction Many proteins gain physiological functions by folding into their own unique three-dimensional structures, and any disturbance during this folding process potentially disrupts protein functions, which is considered to cause a variety of diseases [bib_ref] Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging, Morimoto [/bib_ref]. Among those, neurodegenerative diseases have been well characterized by abnormal accumulation of "mis"-folded proteins in brains and spinal cords of patients [bib_ref] Unfolding the role of protein misfolding in neurodegenerative diseases, Soto [/bib_ref] [bib_ref] Protein aggregation and neurodegenerative disease, Ross [/bib_ref]. More specifically, certain misfolded proteins form insoluble, fibrillar aggregates that are rich in β-sheet structures, widely known as amyloid [bib_ref] Structure of the cross-beta spine of amyloid-like fibrils, Nelson [/bib_ref]. In many neurodegenerative diseases, neurological symptoms appear in middle age (50 years ∼), suggesting that it is a rare event for proteins to become misfolded/aggregated. In fact, protein fibrillar aggregation in vitro requires a significant conformational conversion of proteins to form oligomers (also known as "nucleus"), which is a rate-limiting step of the overall aggregation reaction [bib_ref] Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and..., Harper [/bib_ref]. Once the nucleus forms, however, it functions as a structural template (or so called "seed") to convert native proteins into β-sheetrich structures and then elongate the protein fibril. This mechanism, which accelerates and even triggers protein aggregation, is called the seeding reaction. While it remains controversial whether protein aggregation is the direct cause or a mere result of neurodegeneration [bib_ref] The two faces of protein misfolding: gain-and loss-of-function in neurodegenerative diseases, Winklhofer [/bib_ref] [bib_ref] Amyloid deposits: protection against toxic protein species?, Treusch [/bib_ref] , this seeding mechanism may explain why many neurodegenerative diseases progress rapidly after the symptoms first appear. One notable example for a seeding reaction is the infectivity of Prion diseases, in which the spread of fibrillar aggregates of prion proteins is considered to be the main cause of neurodegeneration [bib_ref] Novel proteinaceous infectious particles cause scrapie, Prusiner [/bib_ref] [bib_ref] The transcellular spread of cytosolic amyloids, prions, and prionoids, Aguzzi [/bib_ref]. Fibrils of prion proteins are considered to work as infectious agents that can be transmitted between individuals. As exemplified in kuru [bib_ref] Oral transmission of kuru, Creutzfeldt-Jakob disease and scrapie to nonhuman primates, Gibbs [/bib_ref] , eating affected tissues of the disease could introduce fibrillar prion aggregates into a brain of a healthy control as seeds and thereby trigger fibrillation of prion proteins and cause neurodegeneration. While no infectivity between individuals has been reported in neurodegenerative diseases besides prion diseases so far, a seeding phenomenon appears to be common to protein fibrillar aggregates [bib_ref] Protein misfolding, evolution and disease, Dobson [/bib_ref] ; therefore, increasing numbers of researchers have pursued possible roles of seeding reactions in pathologies of neurodegenerative diseases [bib_ref] The transcellular spread of cytosolic amyloids, prions, and prionoids, Aguzzi [/bib_ref] [bib_ref] The seeds of neurodegeneration: prion-like spreading in ALS, Polymenidou [/bib_ref] [bib_ref] Transmissible proteins: expanding the prion heresy, Soto [/bib_ref]. For example, Alzheimer's disease (AD) is characterized by fibrillar aggregation of Aβ peptides in brains [bib_ref] The amyloid hypothesis of Alzheimer's disease: progress and problems on the road..., Hardy [/bib_ref] , and accelerated accumulation of Aβ fibrils has been confirmed in primate and rodent models that are injected with brain homogenates of an AD patient [bib_ref] Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host, Meyer-Luehmann [/bib_ref] [bib_ref] Very long term studies of the seeding of beta-amyloidosis in primates, Ridley [/bib_ref]. Several other pathogenic proteins also forms fibrillar aggregates in vitro, which have been tested for their in vivo seeding activity by being transduced into cultured cells and brains of transgenic mouse model [bib_ref] The transcellular spread of cytosolic amyloids, prions, and prionoids, Aguzzi [/bib_ref] [bib_ref] The seeds of neurodegeneration: prion-like spreading in ALS, Polymenidou [/bib_ref] [bib_ref] Transmissible proteins: expanding the prion heresy, Soto [/bib_ref]. A seeding reaction of protein fibrils is thus considered to play important roles in pathological progression of neurodegenerative diseases, and in this mini review, we will focus upon roles of seeded aggregation of proteins in pathologies of amyotrophic lateral sclerosis (ALS). ## A seeded fibrillation of superoxide dismutase (sod1) as a pathological propagation of amyotrophic lateral sclerosis (als) ALS is a devastating motor neuron disease, mainly caused by abnormal accumulation of inclusions in the spinal cord [bib_ref] Unraveling the mechanisms involved in motor neuron degeneration in ALS, Bruijn [/bib_ref]. Notably, ALS has been known to occur as a focal process, which spreads contiguously throughout upper and lower motor neurons [bib_ref] ALS motor phenotype heterogeneity, focality, and spread: deconstructing motor neuron degeneration, Ravits [/bib_ref] [bib_ref] Amyotrophic lateral sclerosis and organ donation: is there risk of disease transmission?, Holmes [/bib_ref] [bib_ref] Can regional spreading of amyotrophic lateral sclerosis motor symptoms be explained by..., Kanouchi [/bib_ref]. In other words, motor neuron degeneration in ALS is an orderly and actively propagating process, which appears to share characteristics of a seeded aggregation of proteins seen in Prion diseases. Most ALS cases (∼90%) are sporadic with no known genetic factors (sporadic ALS, sALS), while the remaining cases have been known to exhibit a family history (familial ALS, fALS; [bib_ref] The changing scene of amyotrophic lateral sclerosis, Robberecht [/bib_ref]. In 1993, dominant mutations in the gene encoding Cu, Zn-superoxide dismutase (SOD1) were identified as one of major genetic causes of fALS [bib_ref] Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral..., Rosen [/bib_ref] , and mutant SOD1 proteins have been known to accumulate abnormally in the form of insoluble inclusions within affected spinal motor neurons of SOD1-related fALS patients [bib_ref] Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from..., Bruijn [/bib_ref]. Ultrastructural analysis of SOD1-positive inclusions in fALS cases has identified their fibrillar morphologies [bib_ref] New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with..., Kato [/bib_ref] ; however, those inclusions were not stained by amyloiddiagnostic dye, Thioflavin S, which has made it controversial whether fibrillar aggregates of mutant SOD1 in vivo are rich in βsheets [bib_ref] Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of..., Kerman [/bib_ref]. Moreover, SOD1-positive inclusions have never been isolated from fALS cases, so further biochemical tests will be required to characterize pathological SOD1 aggregates. In contrast, SOD1-positive inclusions with ALS-like symptoms were reproduced in a fALS-model mouse expressing human SOD1 with a pathogenic mutation [bib_ref] Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS, Turner [/bib_ref] and were found to be stained by Thioflavin S, supporting the formation of amyloid-like, β-sheet-rich fibrils in mouse [bib_ref] Fibrillar inclusions and motor neuron degeneration in transgenic mice expressing superoxide dismutase..., Wang [/bib_ref] [bib_ref] Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial..., Furukawa [/bib_ref]. Insoluble SOD1 aggregates were also successfully isolated from the spinal cords of affected fALS-model mice, and quite notably, those SOD1 aggregates exhibited seeding activity toward fibrillation of purified SOD1 proteins in vitro. have prepared homogenates of spinal cords of transgenic mice expressing human SOD1 with G93A mutation and shown that the homogenates triggered fibrillation of wild-type as well as G93A-mutant human SOD1 proteins under in vitro conditions with acidic pH of solution in the presence of a chaotropic reagent, guanidine hydrochloride [bib_ref] Superoxide dismutase 1 and tgSOD1 mouse spinal cord seed fibrils, suggesting a..., Chia [/bib_ref]. While destabilization of SOD1 proteins under artificial conditions appears to be required for a seeded acceleration of fibrillar aggregation, inclusions containing mutant SOD1 would function as seeds and thereby contribute to propagation of pathological changes among contiguous motor neurons and then disease progression of SOD1-related fALS cases. Fibrillogenic propensities of SOD1 proteins have been well characterized in in vitro studies using purified recombinant proteins. SOD1 is a cytoplasmic enzyme [bib_ref] Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes, Chang [/bib_ref] that catalyzes the conversion of superoxide radicals to hydrogen peroxide and oxygen [bib_ref] Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein), Mccord [/bib_ref] and is activated by binding of a catalytic copper and a structural zinc ion and also by forming an intramolecular disulfide bond [bib_ref] Oxygen-induced maturation of SOD1: a key role for disulfide formation by the..., Furukawa [/bib_ref]. Wild-type holo-SOD1 with a disulfide bond exhibits high thermostability (T m ∼ 90 - C), conferring significant resistance to structural changes and aggregation [bib_ref] On the stability of bovine superoxide dismutase. The effects of metals, Forman [/bib_ref]. In contrast, when SOD1 lacks both metal ions and a disulfide bond (apo-SOD1 SH ), its melting temperature decreases down to 43 - C and become more prone to misfolding and aggregation at physiological temperature [bib_ref] Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo,..., Furukawa [/bib_ref]. In vitro aggregates of human SOD1 polypeptide without any modifications possess amyloid-like characters with fibrillar morphologies and show a seeding activity to accelerate fibrillation of native human SOD1 proteins [bib_ref] Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial..., Furukawa [/bib_ref]. More importantly, amyloid-like fibrils of human apo-SOD1 SH retain their seeding activity in the intracellular environment; transduction of those human SOD1 fibrils into cultured cells (mouse neuroblastoma, Neuro2a) has been shown to trigger the aggregation of stablytransfected human SOD1 . fALS-causing mutations have been shown to decrease affinity for copper/zinc ions and/or stability of a disulfide bond [bib_ref] Decreased metallation and activity in subsets of mutant superoxide dismutases associated with..., Hayward [/bib_ref] [bib_ref] Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to..., Tiwari [/bib_ref] [bib_ref] Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial..., Furukawa [/bib_ref]. Therefore, in a reducing environment of the cytoplasm with high metal-chelating capacity, pathogenic mutations are supposed to increase intracellular fractions of fibrillationprone apo-SOD1 SH [bib_ref] Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial..., Furukawa [/bib_ref]. Nonetheless, it remains unclear how mutant SOD1 forms aggregates under pathological conditions. To elucidate how SOD1 aggregates form, several pathways for aggregation have been proposed in SOD1 proteins in vitro [bib_ref] Protein aggregates in pathological inclusions of amyotrophic lateral sclerosis, Furukawa [/bib_ref]. As reported by Munch et al. mutant SOD1 was found to form fibrillo-granular aggregates by addition of trifluoroethanol (TFE; [bib_ref] Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants, Münch [/bib_ref] , which penetrated inside neuronal cells through macropinocytosis and then acted as seeds to trigger intracellular aggregation of endogenously expressed SOD1 variants [bib_ref] Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells, Münch [/bib_ref]. Once SOD1 aggregation occurs in a cell, the aggregates can be released to the extracellular space and then transferred from cell to cell (vide infra). Intracellular aggregation of mutant SOD1 is thus considered to be persistent and heritable after passages, supporting prion-like propagation of aggregation phenotypes. As mentioned above, two distinct types of SOD1 aggregates, i.e., apo-SOD1 SH amyloids and TFE-induced aggregates, have been found to function as seeds to trigger SOD1 aggregation intracellularly, but their structural and biochemical properties depend on how aggregation was induced [bib_ref] Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial..., Furukawa [/bib_ref] [bib_ref] Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants, Münch [/bib_ref]. Based upon previous in vitro studies, several distinct pathways for aggregation are possible in SOD1 [bib_ref] Disulfide scrambling describes the oligomer formation of superoxide dismutase (SOD1) proteins in..., Toichi [/bib_ref] and are expected to produce SOD1 aggregates with a varying degree of a seeding activity. This might describe heterologous progression and severity of diseases among SOD1related fALS patients. Indeed, disease phenotypes of fALS cases have been known to be variable among different mutations in SOD1 [bib_ref] Protein aggregation and protein instability govern familial amyotrophic lateral sclerosis patient survival, Wang [/bib_ref] , and furthermore, mutation-dependent structures of SOD1 fibrils closely correlate with their distinct biochemical properties [bib_ref] Mutationdependent polymorphism of Cu,Zn-superoxide dismutase aggregates in the familial form of amyotrophic..., Furukawa [/bib_ref]. Therefore, it will be interesting to test if SOD1 fibrils with different mutations exhibit distinct activity as seeds in vitro and in vivo. ## Propagation of protein misfolding in amyotrophic lateral sclerosis (als) In a seeding reaction, sheared pieces of insoluble fibrils can act as structural templates for a "phase-like transition" from soluble native conformers to generally insoluble fibrillar state, but this view now appears to be necessary for revision. antibodies (3H1 and 10C12) that exclusively recognize misfolded SOD1 with disease-specific epitopes, which are not available in the natively folded state, and showed that misfolding of endogenous wild-type SOD1 in human cells (e.g., human embryonic kidney 293 cells (HEK293)) is induced by co-expression of a soluble misfolded form of human SOD1 with pathogenic mutations [bib_ref] Intermolecular transmission of superoxide dismutase 1 misfolding in living cells, Grad [/bib_ref]. In other words, soluble misfolded conformers of SOD1 are also transmissible without adopting classical, insoluble fibrillar states. Furthermore, transient expression of mutant human SOD1 in murine cells (e.g., Neuro2a) did not induce misfolding of endogenous mouse wild-type SOD1 [bib_ref] Intermolecular transmission of superoxide dismutase 1 misfolding in living cells, Grad [/bib_ref]. The difference lies in the amino acid sequence of murine and human SOD1, where the only tryptophan in human SOD1 (Trp32) is replaced by serine in murine counterpart. Indeed, misfolding of wild-type human SOD1 was observed by human SOD1 with a pathogenic (G127X) mutation but was significantly mitigated when G127X human SOD1 with W32S mutation was used [bib_ref] Intermolecular transmission of superoxide dismutase 1 misfolding in living cells, Grad [/bib_ref]. Trp32 in human SOD1 is highly solventexposed and distant from the native dimer interface, which might provide an alternative site for abnormal intermolecular interactions through hydrophobic interactions. It is interesting to note that expression of mutant TAR DNA binding protein 43 (TDP-43) and Fused in Sarcoma (FUS), pathogenic proteins also known to be found in ALS patients [bib_ref] TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration..., Arai [/bib_ref] [bib_ref] Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, Neumann [/bib_ref] [bib_ref] Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral..., Kwiatkowski [/bib_ref] [bib_ref] Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis..., Vance [/bib_ref] , can increase the immunoreactivity for misfolded SOD1 using a disease-specific antibody (3H1), both in patients and cultured human cells (SH-SY5Y; [bib_ref] Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1..., Pokrishevsky [/bib_ref]. While pathological involvement of wild-type SOD1 in ALS remains to be established, aberrant conformers of wild-type SOD1 have been reported in sporadic ALS with no genetic background [bib_ref] Pathological roles of wild-type Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis, Furukawa [/bib_ref]. Accordingly, toxic conformers of SOD1 might be produced by abnormal interactions of folded SOD1 with misfolded SOD1 or other proteins (such as TDP-43/FUS) at the site surrounding Trp32. In other words, as proposed in the template-assisted misfolding of prion proteins [bib_ref] Deadly conformations-protein misfolding in prion disease, Horwich [/bib_ref] , soluble but misfolded conformers of protein molecules can be propagated through abnormal interactions among homologous proteins even without the formation of classical amyloid-like fibrils. ## A cell-to-cell transfer of intracellular superoxide dismutase (sod1) SOD1 is known as one of major intracellular proteins, and most of SOD1 (∼70%) exist in the cytoplasm [bib_ref] Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes, Chang [/bib_ref]. To confirm that seeded aggregation or misfolding of SOD1 is the key molecular mechanism of pathological propagation of SOD1-fALS, it is required to understand how intracellular misfolded/aggregated SOD1 is transferred from the cytoplasm to extracellular environment. As a relatively simple process, misfolded/aggregated SOD1 would be released to extracellular environment by death of an affected cell and then phagocytosed by the other cell. Recent studies have nonetheless suggested more sophisticated processes for a cell-to-cell transfer of SOD1 proteins [bib_ref] Intermolecular transmission of superoxide dismutase 1 misfolding in living cells, Grad [/bib_ref] [bib_ref] Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells, Münch [/bib_ref]. In fact, active secretion of SOD1 to extracellular space has been suggested in several different types of cultured cells [bib_ref] Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells..., Mondola [/bib_ref] [bib_ref] Secretion and increase of intracellular CuZn superoxide dismutase content in human neuroblastoma..., Mondola [/bib_ref] , and both wildtype and mutant SOD1 can be also detected in the cerebrospinal fluid of healthy controls as well as fALS patients [bib_ref] Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients, Zetterström [/bib_ref]. In conditioned media of mouse motor neuron-like hybrid (NSC-34) cell line, impaired secretion of mutant SOD1 was associated with intracellular formation of inclusions and toxicity, suggesting secretion of mutant SOD1 as a beneficial process for cell survival [bib_ref] Impaired extracellular secretion of mutant superoxide dismutase 1 associates with neurotoxicity in..., Turner [/bib_ref]. In contrast, have found that mutant SOD1 proteins are secreted in association with chromogranins and cause microgliosis and neuron death [bib_ref] Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis, Urushitani [/bib_ref] , leading to the idea that suppression of extracellular mutant SOD1 is a promising strategy for therapeutics of SOD1-related fALS cases. Indeed, passive as well as active immunizations targeting extracellular mutant SOD1 proteins have successfully prolonged lifespan of transgenic mice expressing mutant human SOD1 [bib_ref] Therapeutic effects of immunization with mutant superoxide dismutase in mice models of..., Urushitani [/bib_ref]. Toxic roles of secreted SOD1 are further supported by the findings that motor neurons are killed by being co-cultured with astrocytes derived from adult neural progenitor cells isolated from post-mortem lumber spinal cord tissues from sporadic ALS as well as SOD1-related fALS [bib_ref] Astrocytes from familial and sporadic ALS patients are toxic to motor neurons, Haidet-Phillips [/bib_ref]. Also importantly, suppression of SOD1 in both fALS and sporadic ALS astrocytes was found to negate such toxicity of astrocytes toward motor neurons. Recently, furthermore, Basso et al. have shown the increased release of exosomes from astrocytes overexpressing fALS-causing mutant SOD1 and found that astrocyte-derived exosomes contained mutant SOD1 proteins and were transferred to the cytoplasm of spinal neurons [bib_ref] Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome..., Basso [/bib_ref]. Based upon these results, secretion of SOD1 is considered to occur through several distinct pathways and appears to be a normal physiological process. Experimental evidences are further required to show that secretory vesicles act as a messenger to generate seeding activity of SOD1. More specifically, conformational analysis of SOD1 (folded, misfolded, or fibrillized) included in those vesicles will reveal the molecular mechanism of pathological propagation in ALS through a seeding reaction. ## Summary As briefly summarized above, increasing numbers of recent studies have supported the idea that misfolding/aggregation of mutant SOD1 is transmissible through a seeding mechanism inside the cell and among cells. In that sense, it is interesting to test pathological roles, if any, of SOD3, which resides at the extracellular matrix and possesses a structural domain almost homologous to SOD1 [bib_ref] Extracellular superoxide dismutase (SOD3): tissue-specific expression, genomic characterization, and computer-assisted sequence analysis..., Folz [/bib_ref]. A SOD1-like domain of SOD3 has been shown to exhibit propensities for aggregation [bib_ref] Aggregate formation in Cu,Zn superoxide dismutase-related proteins, Son [/bib_ref] , implying its involvement in the formation of seeds that can be taken up by cells. In summary, a seeded aggregation of SOD1 proteins including wild-type SOD1 will be a key event to understand progression/propagation of pathological changes in SOD1-related fALS and even sALS cases without mutations in Frontiers in Cellular Neuroscience www.frontiersin.org March 2014 | Volume 8 | Article 83 | 3 SOD1, and extracellular SOD1 with aberrant conformations is a promising target for therapeutics of those devastating diseases. ACKNOWLEDGMENTSThis work was supported by Grants-in-Aid 24111542 for Scientific Research on Innovative Areas, 25291028 for Scientific Research (B), and 24657093 for Challenging Exploratory Research (to Yoshiaki Furukawa) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

COVID-19 and Cardiac Arrhythmias: a Contemporary Review Purpose of Review A significant proportion of patients infected by the severe acute respiratory syndrome-coronavirus (SARS-CoV2) (COVID-19) also have disorders affecting the cardiac rhythm. In this review, we provide an in-depth review of the pathophysiological mechanisms underlying the associated arrhythmic complications of COVID-19 infection and provide pragmatic, evidence-based recommendations for the clinical management of these conditions. Recent Findings Arrhythmic manifestations of COVID-19 include atrial arrhythmias such as atrial fibrillation or atrial flutter, sinus node dysfunction, atrioventricular conduction abnormalities, ventricular tachyarrhythmias, sudden cardiac arrest, and cardiovascular dysautonomias including the so-called long COVID syndrome. Various pathophysiological mechanisms have been implicated, such as direct viral invasion, hypoxemia, local and systemic inflammation, changes in ion channel physiology, immune activation, and auto- # Introduction In December 2019, an outbreak of 27 cases of pneumonia due to the severe acute respiratory syndromecoronavirus (SARS-CoV2) was reported from Wuhan, China, to the China National Health Commission, prompting the World Health Organization (WHO) to issue a Public Health Emergency of International Concern (PHEIC) in March 2020 . Since then, the disease labeled coronavirus 2019 (COVID-19) caused by SARS-CoV2 has reached pandemic proportionsinfecting a total of more than 276 million people as of December 23, 2021, including 5.4 million deaths worldwide (https:// covid 19. who. int) ]. The majority of cases tend to be asymptomatic or associated with mild systemic and respiratory symptoms (fever, cough, fatigue), but a significant minority of patients develop severe symptoms that may lead to ARDS and hypoxemic respiratory failure, shock, multi-organ failure, and death. There are major disparities in care related to racial, ethnic, demographic, geographic, socio-economic, and political factors, resulting in variability in incidence of new infections and access to appropriate healthcare resources for the management of severe cases. Data from the American Heart Association's COVID- [bib_ref] Atrial fibrillation in patients hospitalized with COVID-19: incidence, predictors, outcomes, and comparison..., Musikantow [/bib_ref] Cardiovascular Disease Registry showed that Hispanic and Black patients comprised more than half of the patients hospitalized with COVID-19; these patients were younger; had higher prevalence of underlying conditions such as hypertension, diabetes mellitus, and obesity; were more often uninsured; had lower socio-economic status; and had longer delays from symptom onset to COVID-19 diagnosis. Although Asian patients had the highest risk-adjusted cardiorespiratory disease severity at presentation compared to the other racial subgroups, no significant differences in in-hospital mortality or occurrence of major adverse cardiovascular events were seen between the racial subgroups after adjustment for sociodemographic characteristics, clinical comorbidities, and clinical presentation [3- ]. However, multiple "waves" of COVID-19 in various countries around the world continue to occur, in spite of significant advancements in our understanding of the disease and its management as well as development of an effective vaccine. Due to the sheer magnitude of the pandemic, the ferocity with which the SARS-CoV2 virus mutates leading to several new variants which respond variably to various treatment regimens, and the slow implementation of effective and widespread vaccination strategies worldwide, the disease continues to ravage humanity and take an enormous toll on our collective physical, social, and psychological well-being. The most common extra-pulmonary manifestations of COVID-19 involve the cardiovascular system, and case series early in the evolution of the disease reported that cardiac injury (manifested as elevated cardiac biomarker levels) occurs in 20-30% of hospitalized COVID-19 patients, and that cardiac injury is independently associated with adverse outcomes including mortality . Risk factors associated with increased mortality from COVID-19 include age , African-American ancestry [bib_ref] Genetic susceptibility for COVID-19-associated sudden cardiac death in African Americans, Giudicessi [/bib_ref] [bib_ref] COVID-19 and African Americans, Yancy [/bib_ref] , history of pre-existing cardiovascular disease [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref] , and co-morbid conditions such as hypertension, diabetes mellitus, obesity, heart failure, renal dysfunction, and chronic lung disease [bib_ref] Gopinathannair R, et al. COVID-19 and cardiac arrhythmias: a global perspective on..., Martinez-Rubio [/bib_ref]. Cardiac manifestations of acute COVID-19 infection include acute myocardial infarction, myocarditis leading to cardiomyopathy and potentially cardiogenic shock, bradyarrhythmias including atrioventricular block, and a plethora of supraventricular (SVT) and ventricular arrhythmias (VA) . There is also growing evidence suggesting the association of COVID-19 infection with development of autonomic dysfunction leading to postural orthostatic tachycardia syndrome (POTS) and inappropriate sinus tachycardia (IST). Clinicians caring for patients with severe COVID-19 infection need to be vigilant for the development of cardiac manifestations, with prompt diagnosis and treatment of these complications in conjunction with pulmonary manifestations of the disease in the acute setting, followed by institution of measures to monitor patients following recovery. In this review, we discuss the most common arrhythmic manifestations of COVID-19, which include atrial fibrillation, other supraventricular arrhythmias, bradycardia and atrioventricular (AV) block, VA, and conditions associated with autonomic dysfunction as well as the clinical features of "long COVID-19." We will summarize published guidelines for the management of arrhythmias associated with COVID-19 and provide practical guidance for clinicians caring for COVID-19 patients with these arrhythmic complications in both inpatient and outpatient settings. [fig_ref] Table 1: Summary of the pathophysiology and management of arrhythmias in COVID-19 patients [/fig_ref] summarizes the various pathophysiological mechanisms that are associated with the myriad arrhythmias seen in COVID-19 patients. Both atrial and ventricular arrhythmias can be caused by direct cardiomyocyte invasion [bib_ref] Association of cardiac infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases, Lindner [/bib_ref] and downstream effects such as reduced expression and activity of ACE2 leading to activation of the renin-angiotensin-aldosterone axis and increased angiotensin II levels, as well as activation of cellular immune responses leading to a hyperinflammatory state and increased production of pro-inflammatory cytokines. Critically ill COVID-19 patients may develop a state called cytokine release syndrome (CRS), characterized clinically by rapid deterioration of respiratory status and ARDS, accompanied by signs of multi-organ dysfunction or failure, arterial and venous thromboses, and hemodynamic compromise that can progress to shock. CRS in COVID-19 patients is characterized biochemically by elevated levels of interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 12 (IL-12), monocyte chemoattractant protein 1(MCP-1), and other biomarkers such as ferritin, fibrinogen, C-reactive protein (CRP), D-dimer, procalcitonin, lactate dehydrogenase (LDH), and von Willebrand factor antigen and activity [bib_ref] Clinical characteristics and outcomes of older patients with Coronavirus Disease 2019 (COVID-19)..., Chen [/bib_ref] , which makes them vulnerable to both atrial and ventricular arrhythmias. ## Pathophysiological mechanisms underlying arrhythmic manifestations in covid-19 patients In addition, activation of the immune system leads to reduced Treg lymphocyte number and activity, increased recruitment and activation of CD4 + CD28-null T cells and differentiation into Th1 subtype of helper T cells, and increased sympathetic activation (both at the central and peripheral levels) leading to increased catecholamine levels, further increasing the potential for atrial and ventricular tachyarrhythmias. Atrial fibrillation can also occur as a result of hypoxemia, acute changes in pulmonary artery and - DC Cardioversion ± initiation of anti-arrhythmic agent Stable patients: - Amiodarone (oral/intravenous) - Atrial fibrillation ablation - AV node ablation and pacing Anticoagulation: Use the CHA2DS2-VASc score to determine need - Antithrombin inhibitors (dabigatran) - Factor Xa inhibitors (apixaban, rivaroxaban)avoid in patients on lopinavir/ritonavir or tocilizumab - Warfarin ## Arrhythmia type Pathophysiological mechanisms ## Management Ventricular arrhythmias [bib_ref] Arrhythmias and COVID-19: a review, Dherange [/bib_ref] [bib_ref] Cardiovascular manifestations of COVID-19 infection, Magadum [/bib_ref] [bib_ref] Cardiac arrhythmias in critically ill patients with COVID-19: a brief review, Karamchandani [/bib_ref] [bib_ref] Cardiovascular disease in patients with COVID-19: evidence from cardiovascular pathology to treatment, Luo [/bib_ref] - Myocarditis leading to a hyperinflammatory state with prolongation of ventricular action potential duration (IL-1, IL-6, TNF-alpha affecting KCNH2/hERG channel function) - Hypoxia causing pathological increases in late sodium currents via the SCN5A Nav 1.5 channels and increased ventricular action potential duration, increased extracellular K + levels reducing depolarization threshold, reduced electrical coupling and more tissue anisotropy due to altered connexin 43 function - Prolonged repolarization and reduced conduction velocity leading to triggered ectopy due to after-depolarizations and re-entry - Cytochrome system inhibition (e.g., IL-6 inhibiting CYP3A4, or drugs such as lopinavir/ ritonavir) causing altered drug metabolism of QT-prolonging medications such as azithromycin, hydroxychloroquine (and statins) and increased risk of torsade de pointes (and rhabdomyolysis) - Myocardial cell death and fibrosis/scar formation, promoting scar-mediated re-entrant arrhythmias (direct cytopathic effects, microvascular dysfunction and micro-thrombi, large-vessel thrombosis causing acute myocardial infarction) Unstable patients: - Electrical cardioversion/ defibrillation ± initiation of anti-arrhythmic agent Stable patients: Monomorphic VT: - IV amiodarone - IV lidocaine - IV procainamide Polymorphic VT (no QT prolongation): - IV lidocaine - IV sedation, anxiolytics Torsade de pointes: - IV isoproterenol - IV magnesium - Temporary pacing - Stop QT-prolonging meds Refractory VT/VT storm: These mechanisms may also contribute to other tachyarrhythmic manifestations such as ventricular arrhythmias right ventricular hemodynamics from acute pulmonary embolism or cor pulmonale, changes in atrial wall compliance and stiffness due to microvascular dysfunction and changes in atrial perfusion and contractility, and in later stages, development of atrial fibrosis. Patients with COVID-19-induced CRS may develop Atrial fibrillation (AF) (de novo or recurrence of pre-existing PAF) due to the impact of cardiorespiratory compromise on intra-cardiac hemodynamics and/or electrophysiologic properties of atrial cardiomyocytes, as well due to the development of a viral-mediated inflammatory atrial cardiomyopathy. This inflammatory cardiomyopathy is characterized by lymphocytic infiltration within the atrial cardiomyocytes, myocardial necrosis, and microangiopathic changes within the atrial vasculature. In addition, direct viral invasion and lymphocytic infiltration of the ganglionated right atrial plexi have been reported, which may predispose to the development of AF as well as sinus node dysfunction (discussed later in this paper). Finally, endothelial cell activation, as well as the activation of various elements of the clotting cascade, can lead to widespread thromboses within the pulmonary vessels (both arterial and venous), as well as in atypical locations such as the right atrial appendage [bib_ref] COVID-19-induced cytokine release syndrome associated with pulmonary vein thromboses, atrial cardiomyopathy, and..., Goette [/bib_ref]. [formula] - IV amiodarone - IV lidocaine - Deep sedation - VT ablation [/formula] Other pathophysiological mechanisms can contribute to the development of VA and sudden cardiac arrest (SCA) in COVID-19 patients. These include ischemia due to thrombosis of small vessels within the myocardium and consequent changes in ion channel function and metabolic changes in ischemic myocytes, and QT interval prolongation both due to increased IL-6 levels and the use of QT-prolonging medications [bib_ref] Plasma concentrations of haloperidol and prolactin and clinical outcome in acutely psychotic..., Aschauer [/bib_ref]. Other important considerations that affect the risk of VAs in COVID-19 include the following: hepatic and renal insufficiency that can cause electrolyte derangements such as hyperkalemia as well as affect metabolism and excretion of QT-prolonging medications; hyperadrenergic state related to critical illness, anxiety, or agitation due to respiratory difficulties; disruption of the sleep-wake cycle and aggravation of pre-existing sleep disorders; nutritional deficiencies due to reduced appetite, prolonged ICU hospitalization, and intubated status; and the use of antimicrobials that can cause QT prolongation either individually or in combination with other antimicrobials or medications used to treat other co-morbid conditions. Of note, most fatal cardiac events in COVID-19 patients are due to non-shockable rhythm disorders such as asystole or pulseless electrical activity, rather than VAs [bib_ref] Management of arrhythmias associated with COVID-19, Desai [/bib_ref]. Various pathophysiologic mechanisms have been proposed to explain the autonomic abnormalities seen in acutely ill COVID-19 patients as well as long COVID syndromes. There is renewed understanding of the interplay between the sympathetic and parasympathetic nervous systems and other neuroendocrine systems including the hypothalamus-pituitary-adrenocortical axis, the renin-angiotensin-aldosterone system, and the arginine-vasopressin system. During the acute phase of COVID-19, hypovolemia due to fever, reduced oral intake due to anorexia or nausea, and diaphoresis, along with deconditioning from prolonged bed rest and immobility, can lead to enhanced sympathetic outflow through the adrenergic and noradrenergic axes, with subsequent development of tachycardia, exertional dyspnea and intolerance, and generalized fatigue. Direct viral invasion of the medullary centers controlling central sympathetic outflow, as well as invasion of extracardiac postganglionic sympathetic neurons, may also lead to chronic increases in sympathetic outflow. There is increasing evidence that patients with COVID-19 have increased levels of autoantibodies directed against beta 1-adrenergic receptors as well as muscarinic cholinergic receptors, which could cause receptor dysregulation and increased sympathetic outflow as well as reduction in parasympathetic outflow [bib_ref] The possible association between COVID-19 and postural tachycardia syndrome, Goldstein [/bib_ref]. ## Specific arrhythmic manifestations of covid-19 Atrial fibrillation/flutter (AFL) AF is a commonly encountered arrhythmia in critically ill patients, in patients post cardiac surgery, and in hospitalized patients with risk factors for AF including advanced age, history of hypertension, diabetes mellitus, coronary artery disease, or congestive heart failure, among others. There is considerable evidence linking AF with states of systemic inflammation as evidenced by raised levels of inflammatory markers (CRP, IL-6, and tumor necrosis factor-α [TNF-α]) [bib_ref] Association of inflammatory factors with occurrence and recurrence of atrial fibrillation: a..., Wu [/bib_ref]. COVID-19 infection has been shown to cause increased levels of both CRP and IL-6, and therefore, it is not surprising that the incidence of AF is significantly higher in critically ill COVID-19 patients. In a recent retrospective study of 3970 hospitalized patients with reverse transcriptase polymerase chain reaction (RT-PCR)-confirmed COVID-19 infection, the incidence of AF/AFL was 10% (13% in a manually analyzed subset), and 4% in patients without a prior history of atrial arrhythmias. Another study of 301 critically ill patients showed that the incidence of new-onset AF was as high as 14.9% [bib_ref] New-onset atrial fibrillation in critically ill patients with coronavirus disease 2019 (COVID-19), Ergun [/bib_ref]. Patients with new-onset AF/AFL were older and had increased serum IL-6 levels and greater myocardial injury (troponin-I levels) [bib_ref] Atrial fibrillation in patients hospitalized with COVID-19: incidence, predictors, outcomes, and comparison..., Musikantow [/bib_ref]. The occurrence of AF and AFL was associated with increased mortality in this [bib_ref] Atrial fibrillation in patients hospitalized with COVID-19: incidence, predictors, outcomes, and comparison..., Musikantow [/bib_ref] and other studies [bib_ref] Cardiac electrophysiology consultative experience at the epicenter of the COVID-19 pandemic in..., Berman [/bib_ref]. A recently published retrospective analysis of hospitalized PCR-confirmed SARS-CoV2 infection in a single healthcare system in New York showed that AF occurred in 17.6% of these patients, of which a majority (65.7%) had new-onset AF. In propensity-matched analysis, AF was independently associated with a higher risk of in-hospital mortality, and new-onset AF was associated with a 56% higher risk of in-hospital mortality compared to patients with a prior history of AF . The management of AF in COVID-19 patients, as in non-COVID AF patients, involves 2 domains: rate and/or rhythm control, and thromboprophylaxis. In hemodynamically unstable patients, acute management includes the use of electrical cardioversion with or without the concomitant initiation of an anti-arrhythmic agent to prevent AF recurrence. In patients with hemodynamically stable AF, a decision between rate control and rhythm control is dictated by the need to restore sinus rhythm using anti-arrhythmic agents versus the risk of pro-arrhythmia in patients who are critically ill or need to stay on other drugs which may alter atrial cellular electrophysiology or cause drug-drug interactions. In general, rate control with medications such as metoprolol or esmolol is preferred due to relatively shorter half-lives, less risk of hemodynamic consequence compared to other agents, less chances of bronchospasm due to beta-1 selectivity, and less risk of drug-drug interactions. Diltiazem or verapamil may be used, but these agents are less desirable as rate control agents in patients with cardiomyopathy, as well as patients on protease inhibitors such as lopinavir/ritonavir which can reduce the metabolism of the non-dihydropyridine calcium channel blockers. Digoxin may also be used for rate control, but serum levels need to be closely monitored in the elderly, those with renal insufficiency, and those on lopinavir/ritonavir which can inhibit p-glycoprotein and cause higher serum digoxin levels. If rhythm control is desirable, then the use of amiodarone (oral or intravenous) can be considered, but close monitoring of QTc interval on ECG, hepatic and renal function, and pulmonary function (as some cases of acute pulmonary toxicity with short-term use of amiodarone have been reported) is needed. Caution should also be exercised in patients on lopinavir/ritonavir which can affect CYP3A4 function and inhibit amiodarone metabolism. The role of tocilizumab, an IL-6 inhibitor, on the occurrence of AF in critically ill COVID-19 patients with CRS, deserves further study. COVID-19 has been shown to induce a hypercoagulable state [bib_ref] COVID-19-associated coagulopathy and antithrombotic agents-lessons after 1 year, Leentjens [/bib_ref] and can predispose patients to venous thromboembolism in the setting of acute COVID-19 infection [bib_ref] Deep vein thrombosis protocol optimization to minimize healthcare worker exposure in coronavirus..., Dua [/bib_ref]. Patients with COVID-19 and AF are thought to have a higher risk of thromboembolic complications, and young critically ill COVID-19 patients were found to have a higher incidence of ischemic stroke [bib_ref] Large-vessel stroke as a presenting feature of COVID-19 in the young, Oxley [/bib_ref]. The optimal use of anticoagulation in hospitalized patients (non-ICU setting and ICU setting) with COVID-19 has recently been summarized [bib_ref] COVID-19-associated coagulopathy and antithrombotic agents-lessons after 1 year, Leentjens [/bib_ref] and incorporated into the recent NICE guidelines (https:// www. nice. org. uk/ guida nce/ ng191/ chapt er/ Recom menda tions). A recently published open-label, randomized clinical trial with combined data from 3 separate clinical trials assessed the impact of therapeutic-dose anticoagulation on hospitalized COVID-19 patients across 393 sites in 10 countries. Among 2219 non-critically ill hospitalized COVID-19 patients, the use of therapeutic-dose anticoagulation (mostly enoxaparin) was associated with a higher probability of survival to hospital discharge with reduced need for ICU-level organ support compared to prophylactic-dose anticoagulation, and this prompted early termination of this study due to superiority of the therapeutic-dose anticoagulation strategy. The authors opined that for every 1000 hospitalized non-critically ill COVID-19 patients, therapeutic-dose anticoagulation would be associated with survival of 40 additional patients until hospital discharge without ICUlevel organ support at the expense of 7 additional major bleeding events, as compared with usual-care thromboprophylaxis [bib_ref] Therapeutic anticoagulation with heparin in noncritically ill patients with COVID-19, Investigators [/bib_ref]. However, in a cohort of 1098 patients hospitalized with severe COVID-19 disease (defined as needing ICU-level care for organ support), the use of therapeutic-dose anticoagulation did not affect the probability of survival to hospital discharge or the number of days free of cardiovascular or respiratory organ support, compared to usualcare pharmacologic thromboprophylaxis [bib_ref] Therapeutic Anticoagulation with Heparin in Critically Ill Patients with Covid-19, Investigators [/bib_ref]. Whether factor Xa inhibitors such as apixaban or rivaroxaban have differential effects on thrombotic complications of COVID-19 compared to heparin or warfarin is currently uncertain and needs further study. The recently completed MICHELLE trial studied the effect of extended thromboprophylaxis using rivaroxaban 10 mg daily versus control post-hospitalization in 320 COVID-19 patients. After a follow-up period of 35 days, the incidence of the composite primary outcome (symptomatic venous thromboembolism [VTE], VTE-related death, bilateral VTE, symptomatic arterial thromboembolism, myocardial infarction, nonhemorrhagic stroke, major adverse limb event, or cardiovascular death) was significantly lower in the rivaroxaban group compared to control (3.14% versus 9.43%, p = 0.03), with no major bleeding events reported in either of the two groups [bib_ref] Rivaroxaban versus no anticoagulation for post-discharge thromboprophylaxis after hospitalisation for COVID-19 (MICHELLE):..., Ramacciotti [/bib_ref]. The role of vorapaxar, an inhibitor of protease-activated receptor 1, shown to reduce activated factor Xa levels and consequently reduce cytokine expression in alveolar epithelial cells in inflammatory lung diseases [bib_ref] The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome, Ranucci [/bib_ref] , also deserves more study for its effect on activation of the clotting cascade in COVID-19 patients with CRS and ARDS. More studies are needed to determine the subgroups of COVID-19 patients who would benefit the most from use of thromboprophylaxis, as well the optimal drugs, doses, and treatment durations for the use of thromboprophylactic agents in COVID-19 patients. The decision to initiate anticoagulation in COVID-19 patients who develop AF should be determined based on their long-term thromboembolic risk assessed by the CHA2DS2-VASc score (OAC recommended for a score of ≥ 2 for men and ≥ 3 for women). Although some have advocated higher doses of therapeutic anticoagulation for COVID-19 patients with AF citing the hypercoagulable milieu and instances of VTE despite standard-dose anticoagulation in these patients, recent studies have questioned this approach, and more research is needed. Currently, no data exist to support a change in the dose of the anticoagulant used, and standard dosage guidelines should be used. Oral anticoagulation using factor Xa inhibitors (apixaban, rivaroxaban) can be used, but caution should be exercised in patients on lopinavir/ritonavir (higher serum drug levels due to reduced drug metabolism) and tocilizumab (lower serum drug levels due to enhanced CYP activity). Dabigatran, an oral antithrombin inhibitor, can be used with the above antimicrobials, but being in capsule form cannot be crushed and given to patients who are intubated and/or receiving enteral or parenteral nutrition. Warfarin should be used with close monitoring of the INR levels due to the risk of multiple pharmacokinetic and/or pharmacodynamic interactions with other medications, as well as altered dietary intake of vitamin K in ill patients. There is currently no evidence to support the prophylactic use of anti-arrhythmic agents in hospitalized patients with COVID-19. Pre-hospitalization medications for the treatment of AF including rate control agents, anti-arrhythmics, and anticoagulation should be continued without interruption if feasible, with pharmaco-vigilance to adjust the dose or modify the agent used based on clinical, biochemical, and electrocardiographic parameters, and phamacokinetic/ phamacodynamic interactions with other drugs used during the hospitalization. Unless clinically indicated, the medications used for long-term AF management should not be changed due to COVID-19 infection, and close post-discharge monitoring and follow-up are needed to determine the impact of COVID-19 on the natural history of AF in each patient. Decisions related to long-term changes in AF management-changes in medications, consideration of catheter ablation of AF, or use of implantable cardiac devices with or without AV nodal ablation-should be deferred to the outpatient setting as much as possible in stable patients, to allow time for recovery from the acute infection and assessment of lingering symptoms of "long COVID syndrome." Based on the published literature, ablation of atrial fibrillation is rarely needed in the acute setting, and established guidelines should guide the choice of ablation in the long-term management of atrial arrhythmias. ## Atrioventricular block and sinus node dysfunction The incidence of AV block in COVID-19 patients varies from 1-12% and is usually transient in nature [bib_ref] Cardiac arrhythmias in patients with COVID-19, Wang [/bib_ref]. These can manifest as high-grade seconddegree AV block, such as Mobitz II second-degree AV block or 2:1 AV block, which may progress to intermittent complete heart block [bib_ref] High-degree atrioventricular block in COVID-19 hospitalized patients, Dagher [/bib_ref] [bib_ref] Prevalence of bradyarrhythmias needing pacing in COVID-19, Akhtar [/bib_ref]. Many of these patients do not have pre-existing cardiovascular disease or abnormalities on prior echocardiograms, and clinicians need to be aware of the association between acute COVID-19 infection and transient AV block. Potential mechanisms for the development of AV block in COVID-19 patients include the following: focal myocarditis or myocardial edema in the region of the AV node or proximal conduction system, electrolyte disturbances, increased vagal activity during periods of endotracheal suctioning and prone positioning, and the effects of drugs including chloroquine (increases Purkinje fiber refractory period and action potential duration, resulting in AV nodal and infra-Hisian conduction disturbance) [bib_ref] Blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes, Sanchez-Chapula [/bib_ref] , hydroxychloroquine, lopinavir/ ritonavir, and azithromycin. As these conduction abnormalities can be transient, management is directed at treatment of the infection, use of intravenous atropine (for transient bradyarrhythmias) or isoproterenol, or insertion of a temporary transvenous pacemaker (in more sustained cases) as needed to allow time for recovery. Development of heart block in COVID-19 patients is thought to be a poor prognostic sign especially in patients with comorbidities such as hypertension and diabetes mellitus [bib_ref] Bradyarrhythmias in patients with COVID-19: marker of poor prognosis?, Chinitz [/bib_ref] [bib_ref] Cardiac arrhythmias in COVID-19 infection, Kochav [/bib_ref]. However, in milder cases of COVID-19 with new-onset AV block early in the course of the illness, close cardiovascular monitoring, discontinuation of AV nodal blocking agents, and treatment of COVID-19 may be enough to allow recovery of AV conduction. Echocardiography to look for new right or left ventricular dysfunction and assessment of pulmonary arterial pressures is reasonable. Ambulatory heart rhythm monitors should be considered after recovery from acute illness to determine the need for further electrophysiologic evaluation and/or permanent pacemaker implantation based on current guidelines. Sinus node dysfunction leading to severe sinus bradycardia has been reported in up to 15% of cases with the SARS-CoV1 infection [bib_ref] Cardiovascular complications of severe acute respiratory syndrome, Yu [/bib_ref] and has also been reported in a few patients with COVID-19 caused by SARS-CoV2 [bib_ref] Bradyarrhythmias in patients with COVID-19: marker of poor prognosis?, Chinitz [/bib_ref]. The SARS-CoV2 virus interacts with the ACE2 receptor to enter the host cell, and the sino-atrial nodal cells are known to have a high degree of ACE2 expression [bib_ref] The angiotensin-(1-7)/Mas receptor axis is expressed in sinoatrial node cells of rats, Ferreira [/bib_ref]. The development of sinus pauses/arrest may be a reflection of the severity of the acute illness and is also a poor prognostic sign. Management of sinus node dysfunction in COVID-19 patients is similar to that for AV block as described above. ## Ventricular arrhythmias The incidence of ventricular arrhythmias in COVID-19 patients, in large, published case series, has ranged from 1.6 to 5.9% [bib_ref] COVID-19 and cardiac arrhythmias, Bhatla [/bib_ref] [bib_ref] Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19), Guo [/bib_ref]. VAs reported in COVID-19 patients include polymorphic ventricular tachycardias including torsade de pointes (TdP), ventricular tachycardia (VT) storm, and ventricular fibrillation. Although the presence of underlying structural heart disease significantly increases the risk of VAs during acute illness, other factors that impact patients with COVID-19 may contribute to the risk of developing VA even in the absence of underlying structural heart disease. Acute myocardial injury in patients with COVID-19, as evidenced by increased cardiac troponin levels, was noted to significantly increase the risk of VA [bib_ref] Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19), Guo [/bib_ref]. Management of VAs in patients with COVID-19 needs to employ a multipronged approach. For hemodynamically stable and unstable VA in patients with COVID-19, standard ACLS protocols should be followed. Contributing factors such as hypoxia, hypovolemia, electrolyte abnormalities such as hypokalemia and hypomagnesemia, and metabolic acidosis should be corrected aggressively. Vasopressor medications, sedation, and neuromuscular blockade should be employed judiciously. Other factors such as volume overload, increased sympathetic tone, and use of pro-arrhythmic drugs should be closely monitored and managed appropriately. Echocardiography to assess biventricular and valvular function should be obtained in all patients with new-onset VAs during COVID-19 infection, especially those with elevated cardiac biomarkers. Beta-blockers and/or anti-arrhythmic drug therapy should be considered for symptomatic nonsustained VA. In patients with polymorphic VT without QT prolongation, underlying myocardial ischemia should be considered as a potential cause. IV beta-blockers or IV lidocaine may be used, in conjunction with intravenous sedation and use of anxiolytics in patients with respiratory distress, agitation, and anxiety. In patients who have QT prolongation and TdP, the use of QT-prolonging drugs should be stopped or modified, and IV magnesium, IV isoproterenol, or temporary transvenous pacing should be used. For patients with VT storm or recurrent or refractory VT, consider use of IV amiodarone and/or lidocaine, along with deep sedation and endotracheal intubation with ventilatory support. There is limited data supporting the use of substrate-based VT ablation in critically ill COVID-19 patients with VT storm and multiple ICD shocks [bib_ref] Ventricular tachycardia storm management in a COVID-19 patient: a case report, Mitacchione [/bib_ref]. Tocilizumab, a monoclonal antibody that blocks the IL-6 receptor and is being used in critically ill COVID-19 patients, can cause QT interval shortening in association with reduction in CRP and cytokine levels and may be beneficial for use in COVID-19 patients with QT prolongation and VAs [bib_ref] Systemic inflammation and arrhythmic risk: lessons from rheumatoid arthritis, Lazzerini [/bib_ref] [bib_ref] Effective treatment of severe COVID-19 patients with tocilizumab, Xu [/bib_ref]. Currently, there is no data supporting the use of prophylactic anti-arrhythmic therapy for the prevention of VAs in hospitalized COVID-19 patients. ## Sudden cardiac arrest A worldwide survey of cardiovascular professionals early in the COVID-19 pandemic reported that cardiac arrest due to VT/VF was seen in 4.8% and pulseless electrical activity (PEA)/asystole was seen in 5.6% of hospitalized COVID-19 patients [10]. In addition, data from Italy [bib_ref] Out-of-hospital cardiac arrest during the COVID-19 outbreak in Italy, Baldi [/bib_ref] and France [bib_ref] Out-of-hospital cardiac arrest during the COVID-19 pandemic, Marijon [/bib_ref] show that the incidence of out-of-hospital cardiac arrest (OHCA) has also increased significantly during the COVID-19 pandemic. The incidence of OHCA increased by up to 200% in the first few weeks of the COVID-19 pandemic in New York City compared to the same period in 2019, with higher rates of PEA (OR 1.99) and asystole (OR 3.5) accounting for the increase in OHCA even though the prevalence of pre-existing illnesses and the percentage of bystander CPR were similar between the two time periods [bib_ref] Characteristics associated with out-ofhospital cardiac arrests and resuscitations during the novel coronavirus..., Lai [/bib_ref]. Even in areas with low rates of COVID-19 infection, the incidence of OHCA was up to 25% higher during the early months of the pandemic. A larger proportion of OHCAs occurred at home, and the rate of bystander CPR was lower than that in the same period in 2019 [bib_ref] Out-of-hospital cardiac arrest response and outcomes during the COVID-19 pandemic, Uy-Evanado [/bib_ref]. Patients with in-hospital VT/VF arrest would need evaluation for underlying structural heart disease (ischemia, myocardial inflammation, cardiomyopathy) and those with no clear transient or reversible causes should be considered for implantable defibrillator implantation for secondary prevention of SCA per current guidelines. Both bradyarrhythmias and tachyarrhythmias may be the cause of SCA in patients with COVID-19. As noted previously, critically ill COVID-19 patients with elevated cardiac troponin are at higher risk for malignant VAs and mortality. In addition, ECG abnormalities such as QRS and QTc prolongation have been observed in patients with COVID-19 pneumonia , and a small study of 63 patients with COVID-19 pneumonia showed that an index of cardiac electrophysiologic balance (iCEB), computed by the QTc/QRS ratio, was significantly higher in the critically ill COVID-19 pneumonia patients, leading to a potentially higher risk of TdP, compared to less severe cases [bib_ref] Electrocardiographic markers of increased risk of sudden cardiac death in patients with..., Alareedh [/bib_ref]. Interestingly, in a cohort of 100 COVID-19 patients (1/3rd needing hospitalization) who underwent cardiac MRI at a median interval of 71 days after the diagnosis of COVID-19, 78% had evidence of cardiac involvement, and 60% had evidence of myocardial inflammation [bib_ref] Outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus..., Puntmann [/bib_ref] independent of the severity and course of illness, presence of pre-existing conditions, or time since diagnosis. However, this was a small sample of patients and performed at a single center and included a large proportion of patients with new or persistent symptoms. In a prospective study of 44 recovered COVID-19 patients, late gadolinium enhancement was observed in 30% of patients, with a scattered lesion distribution localized in the mid-myocardium and/ or sub-epicardium [bib_ref] Cardiac involvement in COVID-19 patients: mid-term follow up by cardiovascular magnetic resonance, Wang [/bib_ref]. A study of 26 elite athletes who had recovered from COVID-19 and underwent cardiac MRI at a median of 32 days since the diagnosis revealed that cardiac abnormalities were seen in 19% of subjects, manifested as myocardial edema and focal mid-myocardial LGE [bib_ref] Cardiac involvement in consecutive elite athletes recovered from Covid-19: a magnetic resonance..., Malek [/bib_ref]. These studies suggest that a significant proportion of patients who have recovered from COVID-19 may still have residual cardiac abnormalities weeks after the initial diagnosis and clinical recovery, and this may in part explain residual symptoms of exertional dyspnea, fatigue, and palpitations seen in the so-called long-haul COVID-19 syndrome. Other factors associated with the observed increase in SCA during the COVID-19 pandemic, especially the alarmingly higher incidence of OHCA, are the shelter-in-place and lockdown restrictions that were imposed during different periods during the pandemic, patients' reluctance to seek medical care due to fear of contracting COVID-19 at their doctor's office or hospital, strained EMS infrastructure due to the increased volume of activations and consequently longer EMS response times, the additional need to have access to and use personal protective equipment for first responders and front-line healthcare personnel, and the diversion of clinical personnel, administrative focus, and other resources from ongoing chronic disease management to controlling the spread of COVID-19. Although the impact of some of these factors has waned in recent months, the emergence of new SARS-CoV2 variants and waves of new COVID-19 cases in different parts of the world, slow and disparate rates of adoption of COVID-19 vaccination, premature loosening of restrictions related to social distancing, use of face masks, and travel pose a looming threat of creating a similar clinical and social environment if the number of cases continues to increase in the weeks and months ahead. ## Cardiac dysautonomias associated with covid-19 A significant minority of COVID-19 patients have symptoms related to autonomic dysfunction, either concomitantly during the acute phase of the illness or, more commonly, up to several weeks after the acute respiratory and constitutional symptoms have resolved. Although the true prevalence of residual symptoms in COVID-19 patients is still unknown, smaller studies have shown that fatigue was reported by more than 50% of patients, dyspnea was reported by about 30%, and chest pain by about 20% of patients up to 4-8 weeks after hospital discharge [bib_ref] Persistent symptoms in patients after acute COVID-19, Carfi [/bib_ref] [bib_ref] Postdischarge symptoms and rehabilitation needs in survivors of COVID-19 infection: a cross-sectional..., Halpin [/bib_ref]. Some COVID-19 patients have symptoms of lightheadedness, orthostatic intolerance, palpitations, and headaches that linger for several weeks beyond the acute phase, and many of these patients receive multiple referrals to subspecialties such as cardiology, neurology, psychiatry, and physiatry. It has also led to the coinage of terms such as "chronic COVID" or "long-haul COVID" within the general public and parts of the medical community, leading to confusion and ambiguity related to diagnosis and management of these syndromes. It is important for clinicians managing COVID-19 patients to be aware of these clinical entities and correlate these symptoms with current or prior SARS-CoV2 infection, and to initiate treatment directed at these symptoms as well as make the appropriate referrals for additional objective testing and management. Retrospective case series show that lightheadedness, orthostatic intolerance, postural tachycardia, and fatigue are the most common symptoms reported by post-COVID syndrome patients . These case series show the patients are generally young, predominantly female, and report residual symptoms for up to 6 months or longer after the acute phase of the illness. In a retrospective study of 20 patients referred for autonomic testing due to persistent cardiac and/or neurologic symptoms after suspected or confirmed acute COVID-19 infection, the use of a 10-min stand test or a tilt table test led to a diagnosis of postural orthostatic tachycardia syndrome (POTS) in 75% of patients, with a minority diagnosed with neurocardiogenic syncope and orthostatic hypotension . In another study, 27 patients with laboratory-confirmed prior SARS-CoV2 infection referred for autonomic testing were subjected to a rigorous battery of tests to assess postganglionic sympathetic sudomotor function, cardiovagal function, cardiovascular adrenergic function, and thermoregulatory function. Eighty-one percent of patients reported symptoms during the head-up tilt table test, of which 63% reported lightheadedness, 26% reported dyspnea, 19% reported chest pain, and 7% reported palpitations. The most commonly diagnosed clinical syndrome was orthostatic intolerance without significant orthostatic tachycardia or orthostatic hypotension which was observed in 41% of patients, while POTS was diagnosed in 22% and mild orthostatic hypotension was seen in 11% [bib_ref] Autonomic dysfunction following COVID-19 infection: an early experience, Shouman [/bib_ref]. In another case series of 11 post-COVID patients (mean age 46 ± 18 years, 9 females), predominant symptoms included palpitations (82%), chest pain (64%), dyspnea (73%), fatigue (82%), or dizziness (27%). Mean time to symptoms from COVID diagnosis was 40 ± 57 days. Two patients (18%) were diagnosed with inappropriate sinus tachycardia (IST), 2 (18.2%) were diagnosed with POTS, and the remaining patients (63.6%) either were still undergoing evaluation or did not meet formal criteria for IST or POTS. Nine patients received medications (beta-blockers in 5, ibuprofen in 2, midodrine and colchicine in 1 each). Six out of 9 patients reported improvement or resolution of symptoms with medical therapy. The American Autonomic Society has published a guidance statement to help clinicians sift through the confusing terminologies and define the scope of long COVID POTS . They endorsed the terminologies used for the different phases of COVID-19 by the NICE guidelines from the UK. According to these guidelines, "long COVID" includes both ongoing symptomatic COVID-19 (presence of symptoms between 4 and 12 weeks after onset) and chronic COVID (symptoms for > 12 weeks without an alternative explanation; https:// www. nice. org. uk/ guida nce/ ng188). In addition to dyspnea, palpitations, and chest pain, other symptoms of long COVID include joint or muscle pain, headache, cognitive impairment ("brain fog"), sleep disturbances, tingling and/or numbness in the hands and feet, dizziness, earache, tinnitus, loss of taste and/or smell, abdominal discomfort or irregularities in bowel function, skin rashes, anxiety, and depression. Patients with chronic medical conditions such as hypertension, congestive heart failure, diabetes mellitus, and chronic kidney disease have increased sympathetic activity, which can contribute to progression of the disease process and related complications. It is therefore plausible that in these patients, further increases in sympathetic outflow caused by hypoxemia and activation of inflammatory pathways from acute COVID-19 can lead to rapid worsening of clinical status and sudden decompensation. Critically ill COVID-19 patients may develop afferent baroreceptor failure in the carotid body, which in combination with changes in central sympathetic outflow from brainstem structures such as the nucleus tractus solitarius (which has high ACE2 expression and therefore vulnerable to direct SARS-CoV2 invasion) can cause lability of blood pressure and sudden hemodynamic compromise [bib_ref] Dysautonomia: an overlooked neurological manifestation in a critically ill COVID-19 patient, Eshak [/bib_ref]. In addition, withdrawal of the vagal parasympathetic outflow, due to viral invasion of the vagal nerve fibers and/or auto-antibody-mediated dysfunction, can blunt the protective anti-inflammatory effects of the vagal pathways, thus accentuating the hemodynamic and neuro-humoral impact of the heightened sympathetic activity in the setting of acute COVID-19 [bib_ref] Potential role of autonomic dysfunction in COVID-19 morbidity and mortality, Rio [/bib_ref]. A recently published review also identifies other putative mechanisms related to the development of long COVID, including T cell dysregulation, tonically increased secretion of autoantibodies by B cells, viral invasion of intestinal cells and persistence of virus within the gastrointestinal tract, and alterations in gut microbiome content. Risk factors for the development of long COVID include female gender, history of anxiety or depression, age > 70 years, and the presence of > 5 clinical symptoms by the end of the first week of onset. Interestingly, most studies show that the severity of acute illness did not correlate with subsequent development of long COVID [bib_ref] Long COVID or post-COVID-19 syndrome: putative pathophysiology, risk factors, and treatments, Yong [/bib_ref] [bib_ref] Persistent fatigue following SARS-CoV-2 infection is common and independent of severity of..., Townsend [/bib_ref] [bib_ref] Prolonged and late-onset symptoms of coronavirus disease 2019, Miyazato [/bib_ref]. Management of patients with long COVID requires a multi-disciplinary approach centered around empowering the patient through reassurance, education and institution of self-management strategies, symptom-directed non-pharmacologic and pharmacologic therapies, appropriate subspecialty referrals for diagnosis and management of clinical entities (POTS, vasovagal syncope, orthostatic hypotension, other neurologic conditions (such as small fiber neuropathy), arrhythmias and other cardiovascular conditions (such as IST or supraventricular tachycardia), and psychiatric illnesses (such as anxiety disorder, depression, or post-traumatic stress disorder)), and a structured physical rehabilitation program designed to overcome the deconditioning associated with protracted illness. The ACC/AHA/HRS guidelines for the management of syncope are a useful guide for clinicians in managing specific symptoms of long COVID-19 [bib_ref] ACC/AHA/HRS guideline for the evaluation and management of patients with syncope: a..., Shen [/bib_ref]. In patients with orthostatic intolerance, it is helpful to establish correlations between symptoms and objective abnormalities on a tilt table test and then provide targeted recommendations to patients to identify the prodromal symptoms and respond with hydration and counterpressure maneuvers (switch to a seated or supine position, tensing of thigh or buttock muscles, sitting with arms and legs crossed, folding arms and leaning forward with the head down, etc.). A useful educational resource for patients is the STARS initiative from the Heart Rhythm Alliance (www. stopf ainti ng. org). Regular, structured exercise using a recumbent bike or swimming is useful for reconditioning. Adequate fluid (2-3 L/day) and salt intake (5-10 g sodium chloride), avoidance of caffeine or alcohol intake, having smaller meals more frequently rather than large meals, avoidance of known triggers such as prolonged standing or warm environments, and use of compression garments are all measures that can help prevent or alleviate orthostatic symptoms. If medications are used, they should be directed at specific symptoms and/or specific co-morbid conditions. In POTS patients, medications such as fludrocortisone can be used judiciously for patients with hypovolemia, while midodrine may be helpful if orthostatic hypotension is a predominant manifestation. In patients with hyperadrenergic states, clonidine, methyldopa, or propranolol may be used, with close monitoring for adverse effects and rebound hypertension due to sudden discontinuation [bib_ref] Autonomic dysfunction in 'long COVID': rationale, physiology and management strategies, Dani [/bib_ref]. # Conclusions A variety of arrhythmic manifestations have been described in patients with COVID-19, which range from relatively benign conditions such as transient sinus bradycardia to potentially life-threatening conditions such as ventricular tachyarrhythmias and sudden cardiac death. Atrial fibrillation is the most common arrhythmia seen in acutely ill COVID-19 patients. While the pathophysiological mechanisms underlying these arrhythmias in COVID-19 patients are incompletely understood, direct viral invasion, hypoxemia, activation of systemic inflammatory systems with downstream release of cytokines and inflammatory and pro-fibrotic mediators, changes in ion channel physiology, activation of the immune system, and dysregulation of autonomic function have been implicated. Management of these arrhythmias should be based on published evidence-based guidelines, with special consideration of the acuity of COVID-19 infection, concomitant use of antimicrobial and anti-inflammatory drugs, and the transient nature of some arrhythmias. The development of atrial or ventricular arrhythmias in hospitalized COVID-19 patients has been shown to portend a higher risk of in-hospital death. Some manifestations, such as the "long COVID syndrome," may lead to residual symptoms several months after acute infection and need more clinical study and specific management strategies. Finally, as the pandemic evolves with the discovery of new SARS-CoV2 variants, newer drugs for treatment of acute COVID-19 infection, and the widespread administration of vaccines, clinicians must remain vigilant for other arrhythmic manifestations that may occur in association with this novel but deadly disease. ## Compliance with ethical standards ## Conflict of interest [table] Table 1: Summary of the pathophysiology and management of arrhythmias in COVID-19 patients [/table]

Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3.Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription. T he mammalian genome encodes many long noncoding RNAs (lncRNAs) that play pivotal roles in gene regulation during development and disease pathogenesis [bib_ref] Genome regulation by long noncoding RNAs, Rinn [/bib_ref] [bib_ref] Long non-coding RNAs: new players in cell differentiation and development, Fatica [/bib_ref] [bib_ref] The emergence of lncRNAs in cancer biology, Prensner [/bib_ref]. LncRNAs can regulate gene expression through transcriptional interference (TI) [bib_ref] Gene regulation by the act of long non-coding RNA transcription, Kornienko [/bib_ref] [bib_ref] Regulation of transcription by long noncoding RNAs, Bonasio [/bib_ref] or through the RNA product itself [bib_ref] Genome regulation by long noncoding RNAs, Rinn [/bib_ref] [bib_ref] Long non-coding RNAs: new players in cell differentiation and development, Fatica [/bib_ref]. TI occurs when the act of transcribing a gene directly interferes with the transcription of an adjacent gene in cis, either at its initiation site or at an essential cis-regulatory element [bib_ref] Transcriptional interference--a crash course, Shearwin [/bib_ref] [bib_ref] Gene regulation by antisense transcription, Pelechano [/bib_ref]. Transcription of lncRNA has been shown to interfere with the transcription of adjacent genes by altering the binding of transcription factors [bib_ref] Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis..., Petruk [/bib_ref] [bib_ref] Antisense transcription controls cell fate in Saccharomyces cerevisiae, Hongay [/bib_ref] [bib_ref] Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating..., Bumgarner [/bib_ref] and/or RNA polymerase II (Pol II) [bib_ref] Airn transcriptional overlap, but not its lncRNA products, induces imprinted Igf2r silencing, Latos [/bib_ref] , nucleosome remodelling 12 or inducing changes in histone modifications [bib_ref] Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S...., Camblong [/bib_ref] [bib_ref] A ncRNA modulates histone modification and mRNA induction in the yeast GAL..., Houseley [/bib_ref]. So far, the strategies for demonstrating TI in mammalian cells have relied upon genetic engineering methodologies that remove the promoter region or insert premature transcriptional termination sites within the transcriptional unit [bib_ref] Gene regulation by the act of long non-coding RNA transcription, Kornienko [/bib_ref]. An alternative approach would be to leave the DNA template intact and directly target the lncRNA with small interfering RNAs (siRNAs). It is widely perceived that RNA interference (RNAi) is a cytoplasmic pathway for post-transcriptional gene silencing [bib_ref] Molecular mechanisms of RNA interference, Wilson [/bib_ref]. However, RNAi-based technology has been used to posttranscriptionally deplete a lncRNA Kcnq1ot1 in the nucleus and demonstrate that this lncRNA product is not required to maintain imprinting of adjacent genes [bib_ref] Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem..., Golding [/bib_ref]. RNAi has also been shown to induce transcriptional gene silencing (TGS) [bib_ref] Small interfering RNA-induced transcriptional gene silencing in human cells, Morris [/bib_ref] [bib_ref] RNAi factors are present and active in human cell nuclei, Gagnon [/bib_ref] [bib_ref] Argonaute and the nuclear RNAs: new pathways for RNA-mediated control of gene..., Gagnon [/bib_ref]. siRNA-directed TGS can lead to epigenetic changes such as DNA methylation and histone methylation at the target promoters [bib_ref] Small interfering RNA-induced transcriptional gene silencing in human cells, Morris [/bib_ref] [bib_ref] The antisense strand of small interfering RNAs directs histone methylation and transcriptional..., Weinberg [/bib_ref] [bib_ref] Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells, Hawkins [/bib_ref] [bib_ref] Short doublestranded RNA induces transcriptional gene silencing in human cancer cells in..., Ting [/bib_ref]. In addition, promoter-targeting siRNAs can induce TGS by blocking the recruitment and the activity of Pol II . In all these cases, Argonaute proteins (AGO1 and AGO2) were shown to be key players of TGS [bib_ref] Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells, Hawkins [/bib_ref] [bib_ref] Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells, Napoli [/bib_ref] [bib_ref] Bidirectional transcription directs both transcriptional gene activation and suppression in human cells, Morris [/bib_ref] [bib_ref] Involvement of argonaute proteins in gene silencing and activation by RNAs complementary..., Chu [/bib_ref] [bib_ref] Involvement of AGO1 and AGO2 in mammalian transcriptional silencing, Janowski [/bib_ref] [bib_ref] Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells, Kim [/bib_ref]. Finally, it has been shown that siRNAs can be used to knockdown small-nuclear ncRNA 7SK [bib_ref] Specific and potent RNAi in the nucleus of human cells, Robb [/bib_ref]. These observations prompted us to investigate whether siRNAs can be used to inhibit lncRNA transcription and to explore the functional consequences of this process. In particular, we postulated that by targeting different regions of the lncRNA, we could uncouple the act of transcription from the function of the transcript. This would enable us to investigate how lncRNAs regulate adjacent genes in cis through TI. As a model, we used the imprinted tumour-suppressor DIRAS3 locus, where we have recently characterized a novel lncRNA known as GNG12-AS1 (ref. [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. GNG12-AS1 is transcribed in an antisense orientation to DIRAS3 and its neighbouring non-imprinted genes GNG12 and WLS [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. We have shown that this lncRNA is allele specifically silenced in cancer cell lines depending on the imprinted state of DIRAS3 (ref. [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. DIRAS3 (also known as ARHI and NOEY2) is a Ras-related imprinted tumour suppressor involved in the inhibition of growth, motility and invasion via several signalling pathways including RAS/MAPK, STAT3 and PI3K [bib_ref] Biochemistry and biology of ARHI (DIRAS3), an imprinted tumor suppressor gene whose..., Yu [/bib_ref]. DIRAS3 is downregulated in 70% of breast and ovarian cancer [bib_ref] NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast..., Yu [/bib_ref] [bib_ref] Loss of the expression of the tumor suppressor gene ARHI is associated..., Wang [/bib_ref] [bib_ref] Low expression of ARHI is associated with shorter progressionfree survival in pancreatic..., Dalai [/bib_ref] , and its loss of expression correlates with cancer progression and metastasis [bib_ref] Loss of the expression of the tumor suppressor gene ARHI is associated..., Wang [/bib_ref] [bib_ref] Low expression of ARHI is associated with shorter progressionfree survival in pancreatic..., Dalai [/bib_ref]. The mechanism responsible for DIRAS3 downregulation to date involves different epigenetic mechanisms and loss of heterozygosity [bib_ref] Biochemistry and biology of ARHI (DIRAS3), an imprinted tumor suppressor gene whose..., Yu [/bib_ref]. We hypothesized that TI by GNG12-AS1 could represent an additional layer of regulating DIRAS3 dosage. Here, we demonstrate that GNG12-AS1 can be transcriptionally silenced with siRNAs complementary to a region proximal to its transcriptional start site (TSS). The transcriptional silencing of GNG12-AS1, which is mediated by AGO2, leads to upregulation of DIRAS3 transcription in cis, indicating a function in TI. In contrast, targeting GNG12-AS1 at the 3 0 end did not affect its nascent transcription, but reduced the lncRNA through post-transcriptional gene silencing and, importantly, did not affect DIRAS3 transcription. We further show different phenotypic effects in cell cycle and migration depending on whether we target the 5 0 or 3 0 end of GNG12-AS1. Altogether, our results demonstrate that strategic targeting of siRNA to different regions of an lncRNA can enable the discrimination between functions related to its active transcription and that of the RNA product. # Results GNG12-AS1 is a stable lncRNA localized in the nucleus. In this study, we used three non-cancer cell lines (HB2, HS27, MCF10A), which we have previously shown to have normal imprinted DIRAS3 expression, and breast cancer cell lines (SUM159, MCF7), where loss of DIRAS3 imprinting leads to biallelic expression (SUM159) or biallelic silencing of DIRAS3 (MCF7). The non-cancer cell lines expressed GNG12-AS1 from both alleles, whereas the cancer cell lines expressed GNG12-AS1 from one allele [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. We confirmed the relative expression of DIRAS3, GNG12, GNG12-AS1 and WLS in these cell lines [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. Actinomycin D chase experiments indicate that GNG12-AS1 is a stable lncRNA with a half-life between 20 and 25 h [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. DIRAS3 expression remained unchanged in HB2 and increased in HS27 when cells were treated with Actinomycin D, suggesting an inverse relationship between DIRAS3 and GNG12-AS1 transcription. Despite being a stable lncRNA, GNG12-AS1 has a low transcript volume (20-80 molecules per 100 cells; [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. Expression analysis following cell fractionation indicated that GNG12-AS1 is localized within the chromatin [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref] , similar to MALAT1, a lncRNA known to be associated with chromatin [bib_ref] The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites, West [/bib_ref]. Using single-molecule RNA fluorescence in situ hybridization (RNA FISH; as described in ref. [bib_ref] Imaging individual mRNA molecules using multiple singly labeled probes, Raj [/bib_ref] , we confirmed that GNG12-AS1 is nuclear in HB2 and SUM159 cells. Exonic probes complementary to all exons showed that GNG12-AS1 transcripts accumulate in the nucleus in discrete foci in 19% of SUM159 and 25% of HB2 cells [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. Intronic RNA FISH probes complementary to the first intron of GNG12-AS1 were found to co-localize with the exonic signals in about two-thirds of cases where both probes were present in the same cell (6.8% in HB2 cells; 2.8% in SUM159 cells; [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. As intronic probes are usually indicative of nascent transcription and exonic probes can detect both primary and mature transcripts, their co-localization may indicate that the processed transcript remains at its site of transcription, which would fit with GNG12-AS1 being co-transcriptionally spliced, as previously reported [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. However, as we also see separate signals for exonic and intronic probes, it is likely that some GNG12-AS1 also accumulate at other sites in the nucleus away from its site of transcription (3.3% in HB2 cells; 1.7% in SUM159 cells; [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. A caveat to the interpretation of FISH data is that intronic and exonic RNA FISH probes might not hybridize to nascent and mature lncRNA transcripts in a mutually exclusive way. Nascent RNA contains exons and the mature lncRNAs may be present as unspliced isoforms. In SUM159 cells, the intronic probe mostly produced a single FISH signal (13% of cells being monoallelic, [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref] , which fits with previous pyrosequencing data reporting allele-specific GNG12-AS1 expression in cancer cells [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. By contrast, in HB2 cells, even though pyrosequencing showed an allelic ratio of 50% (ref. [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref] , we observed cells with both single and double intronic signals (8 and 6% of cells, respectively; [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref] , suggesting that GNG12-AS1 is heterogeneously biallelic and randomly monoallelic at the cellular level in HB2 cells. Together, these data confirm that GNG12-AS1 is a stable lncRNA that is present in the nucleus at distinct foci. TI between GNG12-AS1 and DIRAS3. To investigate the role of GNG12-AS1, we depleted GNG12-AS1 using siRNAs [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref]. GNG12-AS1 has multiple isoforms 31 , thus we designed siRNAs against several different exons and found that siRNA targeted to exons 1, 5 and 7 reduced GNG12-AS1 by 50-70% in HB2 and SUM159 cells [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref]. These exons are common to all of the known splice forms of GNG12-AS1. Using several primer sets to capture the various GNG12-AS1 isoforms, we confirmed that siRNAs against exons 1 and 7 efficiently knockdown GNG12-AS1 [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref]. We further confirmed that the knockdown occurred in all cellular compartments [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref]. Thus, we are able to deplete GNG12-AS1 in the nucleus with siRNA. RNA FISH analysis in SUM159 cells showed that although siRNAs to either exon 1 or exon 7 effectively ablated GNG12-AS1 detectable with exonic probes, siRNA targeting exon 1 reduced GNG12-AS1 detectable with intronic probes (nascent transcripts) more efficiently than siRNA to exon 7 [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref]. These results indicate that although both siRNAs can efficiently reduce GNG12-AS1 levels in different cellular compartments, siRNA complementary to the 5 0 end of GNG12-AS1 may preferentially inhibit its nascent transcription. We next asked whether depletion of GNG12-AS1 affects the transcription of its neighbouring genes. We analysed the expression of GNG12, DIRAS3 and WLS after depleting GNG12-AS1 with siRNAs against exon 1 and exon 7. DIRAS3, but not GNG12 or WLS, was upregulated after targeting exon 1 of GNG12-AS1 in four cell lines [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref] ,d and [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref]. DIRAS3 upregulation was consistent in different cell lines when GAPDH [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref] ,d and [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref] or the geometric mean of GAPDH and RPS18 were used (Supplementary [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref]. Thus, for further quantitative realtime PCR (qRT-PCR) experiments, we used GAPDH as the reference gene. In a fifth cell line MCF7, despite a 60% reduction in GNG12-AS1, expression of DIRAS3 could not be reactivated [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. In MCF7 cells, DIRAS3 is more hypermethylated at its promoter compared with SUM159 cells [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. Off-target effects were excluded by the use of additional siRNAs against exon 1 and exon 7 of GNG12-AS1 [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. Furthermore, we used the randomized nucleotide sequence (scrambled siRNAs) of exon 1 and exon 7 siRNAs [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref] , as well as C911 mismatch siRNA controls [bib_ref] C911: A bench-level control for sequence specific siRNA off-target effects, Buehler [/bib_ref] , all of which failed to affect GNG12-AS1 and DIRAS3 levels [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. To exclude the possibility of GNG12-AS1 regulating DIRAS3 expression in trans, we ectopically introduced GNG12-AS1 in SUM159 and HB2 cells. Overexpression of the most common splice variants of GNG12-AS1 had no effect on expression of DIRAS3 or the surrounding genes [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. As DIRAS3 is an imprinted gene, we examined whether allelespecific expression changed after GNG12-AS1 depletion. DIRAS3 imprinting and its methylation status were unaltered (Supplementary [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. Taken together, these results suggest that GNG12-AS1 modulates the expression of the already active DIRAS3 allele in cis and that siRNA to exon 1 of GNG12-AS1 disrupts this function without affecting expression of neighbouring genes. siRNA against 5 0 end of GNG12-AS1 blocks RNA Pol II. Next, we examined whether inhibition of DIRAS3 in cis can be achieved **** siRNA: Con ex1 ex7 Con ex1 ex7 Intronic probes Exonic probes SUM159 Con ex1 ex7 Con ex1 ex7 Con ex1 ex7 Con ex1 ex7 siRNA: Con ex1 ex7 Con ex1 ex7 Conex1 ex7 Con ex1 ex7 Con ex1 ex7 by targeting GNG12-AS1 exons closer to the DIRAS3 gene. Similar to when we targeted exon 7, siRNA to exon 5 efficiently reduced GNG12-AS1 [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref] , but did not upregulate DIRAS3 [fig_ref] Figure 6 |: GNG12-AS1 regulates cell migration independently of DIRAS3 [/fig_ref]. In one of the cell lines, siRNA to exon 5 led to WLS downregulation. siRNAs to exons 2 and 3, which flank DIRAS3 on either side, reduced GNG12-AS1 isoform specifically [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref] , suggesting that these siRNAs function post-transcriptionally. These siRNAs had no effect on DIRAS3 expression further supporting modulation via the act of transcription rather than through a specific GNG12-AS1 isoform [fig_ref] Figure 6 |: GNG12-AS1 regulates cell migration independently of DIRAS3 [/fig_ref]. To prove that the cis function of GNG12-AS1 is modulated by siRNA targeting exon 1, which leads to the inhibition of TI between GNG12-AS1 and DIRAS3, we profiled Pol II and histone modifications. As shown in [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref] ,b, a significant reduction in Pol II binding was observed at the TSS of GNG12-AS1 when exon 1, but not exon 7, was targeted by siRNA. Concomitantly, an increase in Pol II binding was observed at the DIRAS3 TSS. Nuclear run-on assays showed that the reduction in Pol II binding was associated with reduced nascent GNG12-AS1 transcription. Thus, a reduction of GNG12-AS1 and increased DIRAS3 run-on transcripts were observed with siRNA targeting exon 1, but not exon 7 [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref]. To determine whether we could ablate unspliced transcripts and if so, whether this could affect DIRAS3 expression, we designed siRNAs to the first intron at 195 and 2,933 bp downstream of the GNG12-AS1 TSS. In both HB2 and HS27 cell lines, these siRNAs resulted in a reduction of GNG12-AS1, indicating that the unspliced GNG12-AS1 transcript can be targeted by siRNA. However, increased DIRAS3 expression occurred only in HS27 cells [fig_ref] Figure 7 |: Inhibition of transcriptional interference with siRNA [/fig_ref] , indicating that siRNA specifically targeting exon 1 is more efficient at disrupting the regulatory relationship between GNG12-AS1 and DIRAS3 transcription. Consistent with the changes in Pol II binding, we observed changes in active histone modifications when GNG12-AS1 was depleted using siRNA against exon 1, but not with exon 7 [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref]. Histone H3 lysine 4 trimethylation (H3K4me3) was reduced at the GNG12-AS1 TSS and increased at the DIRAS3 TSS. Furthermore, histone H3 lysine 36 trimethylation (H3K36me3), a marker for Pol II elongation [bib_ref] Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to..., Krogan [/bib_ref] , was decreased in the body of GNG12-AS1 and increased within DIRAS3. As GNG12-AS1 depletion with exon 1 siRNA did not affect WLS expression, a reduction in H3K36me3 at 3 0 end of GNG12-AS1 correlates with reduction in its transcriptional activity. The reduction of active histone modifications at the GNG12-AS1 TSS was not accompanied by a reciprocal increase of silencing modifications previously reported to be involved in TGS [bib_ref] The antisense strand of small interfering RNAs directs histone methylation and transcriptional..., Weinberg [/bib_ref] [bib_ref] Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells, Hawkins [/bib_ref] [bib_ref] Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells, Kim [/bib_ref] , such as histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 27 trimethylation (H3K27me3; . In addition, DNA methylation was unchanged at the GNG12-AS1 TSS . The changes in Pol II binding at the TSS of GNG12-AS1, together with the changes in active histone modifications associated with transcriptional activity after siRNA to exon 1, suggest that targeting the 5 0 end of this lncRNA results in transcriptional silencing. However, to date, in all instances of TGS with exogenous siRNAs in human cells, the siRNAs were directed to the gene promoters. We therefore designed two siRNAs targeting GNG12-AS1 upstream of the TSS (33 and 129 bp upstream of TSS). These siRNAs had no effect on GNG12-AS1 expression [fig_ref] Figure 7 |: Inhibition of transcriptional interference with siRNA [/fig_ref] , suggesting that transcription needs to be initiated for effective knockdown of the lncRNA. In summary, siRNA targeting specifically exon 1 of GNG12-AS1 reduced its nascent transcription, without inducing changes in silent chromatin marks. Nevertheless, reduced transcription was associated with diminished Pol II activity and the redistribution of active histone marks, which led to increased DIRAS3 expression. AGO2 mediates transcriptional inhibition of GNG12-AS1. As TGS is achieved through the RNAi machinery 24,25,27,28 , we investigated whether siRNA-mediated silencing of GNG12-AS1 involves Argonaute proteins. First, we examined whether the reduction in GNG12-AS1 transcript by siRNA targeted to exon 1 could be rescued by depletion of AGO1/2 proteins. We found that depletion of AGO2 rescued GNG12-AS1 expression in cells exposed to siRNAs against exon 1 and exon 7 and abolished DIRAS3 upregulation in cells treated with siRNA against exon 1 [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. This effect was specific to AGO2, as AGO1 depletion did not rescue GNG12-AS1 depletion or DIRAS3 levels . Similar results were obtained in the HS27 cell line , confirming that AGO2 is involved in both the transcription-and post-transcriptionmediated mechanism of siRNA-directed downregulation of GNG12-AS1. . Expression levels of AGO2, AGO1, DIRAS3 and GNG12-AS1 were normalized to GAPDH and compared with control siRNA by qRT-PCR. AGO1 levels were not affected by either single AGO2 siRNA or double knockdown of AGO2 and GNG12-AS1. Transcriptional upregulation of DIRAS3 can be rescued by depletion of AGO2 and siRNA targeting exon 1 of GNG12-AS1, whereas siRNA-mediated reduction of GNG12-AS1 with both siRNAs expression can be rescued by depletion of AGO2. (b) AGO2 ChIP analysis in HB2 cells after siRNA depletion of GNG12-AS1. The x axis shows enrichment of AGO2 at regions described in [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref]. AGO2 binding was enriched only at the TSS of GNG12-AS1 (a) after siRNA to exon 1. The GAPDH TSS was used as negative control region for AGO2 ChIP. (c) RIP from nuclear HB2 extracts showing association of AGO2 with GNG12-AS1 transcript after treatment of cells with exon 1 but not exon 7 siRNA. U1 and GAPDH were used as negative control RNAs for AGO2 RIP. RIP enrichments are presented as % of input RNA. For all the graphs, error bars indicate s.e.m. (n ¼ 3 biological replicates). *Po0.05 and **Po0.01 by two-tailed Student's t-test. [formula] Control siRNA Exon1 siRNA Exon7 siRNA GAPDH b c d e f g a Relative AGO2 enrichment (% of input) 0 2 AGO2 DIRAS3 GNG12-AS1 -A2 Control - Exon1 - Exon7 A2 A2 AGO1 siRNA: -A2 Control - Exon1 - Exon7 A2 A2 -A2 Control - Exon1 - Exon7 A2 A2 -A2 Control - Exon1 - Exon7 A2 A2 [/formula] We verified that AGO2 is present in the nucleus and cytoplasm , as previously reported [bib_ref] RNAi factors are present and active in human cell nuclei, Gagnon [/bib_ref]. Next, we used chromatin immunoprecipitation (ChIP) analysis to see whether AGO2 associates with the GNG12-AS1 locus. We found that AGO2 was recruited to the GNG12-AS1 TSS only after treatment with siRNA directed to exon 1, but not exon 7 [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. This result suggests that AGO2 facilitates the physical interaction between exogenous siRNA to exon 1 and the chromatin to potentially mediate TGS of GNG12-AS1. Finally, we tested whether GNG12-AS1 associates with AGO2 in the nucleus. RNA immunoprecipitation (RIP) for AGO2 in nuclear extracts transfected with siRNAs showed an interaction between GNG12-AS1 and AGO2 only when siRNA targeted exon 1, and not exon 7 [fig_ref] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1 [/fig_ref]. Taken together, these results demonstrate that AGO2 mediates transcriptional silencing of GNG12-AS1 by siRNA complementary to exon 1. Cell cycle regulation by GNG12-AS1. DIRAS3 has been implicated in cell cycle regulation [bib_ref] Effects of ARHI on cell cycle progression and apoptosis levels of breast..., Li [/bib_ref] , so we next asked if expression levels of DIRAS3 and GNG12-AS1 might correlate at different stages of the cell cycle. We synchronized cells with a double thymidine block [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref] , which arrest cells in S-phase and used cyclin E1 (G1/S transition marker) to monitor cell cycle progression [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. An increase in GNG12-AS1 transcription was observed after the release from double thymidine block, which coincided with the downregulation of DIRAS3. Cell cycle synchronization using serum starvation, followed by release with serum-containing media, showed similar results [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. The inverse relationship between expression levels of GNG12-AS1 and DIRAS3, which was tightly maintained throughout the cell cycle, further supports the evidence that GNG12-AS1 transcription modulates DIRAS3 expression in cis through TI. We next examined the consequence of upregulating DIRAS3 after inhibiting GNG12-AS1. When GNG12-AS1 was depleted with exon 1 siRNA, we observed a small decrease in G1 cell number (control siRNA: 34% versus exon 1 siRNA: 29%; P ¼ 0.0575; [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. This result was confirmed by qRT-PCR and immunoblot analysis, which showed decreased cyclin E1 levels [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. As expected, by combining siRNAs to DIRAS3 and exon 1 siRNA, we were able to neutralize the increase in DIRAS3 expression [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref] , which led to a modest but significant restoration of the G1 population (exon 1 siRNA: 29% versus exon 1/DIRAS3 siRNA: 36%, P ¼ 0.0105; [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. Analysis of cyclin E1 levels showed that the reduction in G1 cell numbers could be partially rescued with the simultaneous depletion of GNG12-AS1 and DIRAS3 [fig_ref] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation [/fig_ref]. By contrast, cyclin B1 levels did not differ among GNG12-AS1-and DIRAS3 siRNA-treated cells. Both RNA and protein levels of the cell cycle inhibitor p21 increased when the cells were treated with siRNA to exon 1 in a DIRAS3-independent manner. S-phase distribution was unchanged after reducing GNG12-AS1. However, both exon 1 and exon 7 siRNAs increased the G2/M population (control siRNA: 23% versus exon 1 siRNA: 35%, P ¼ 0.0009; or versus exon 7 siRNA: 30%, P ¼ 0.0126). As the increase in G2/M population occurred upon simultaneous depletion of GNG12-AS1 and DIRAS3 (control siRNA: 23% versus exon 1/DIRAS3 siRNA: 29%, P ¼ 0.0169; or versus exon 7/DIRAS3 siRNA: 30%, P ¼ 0.0084), we concluded that this G2/M delay is a DIRAS3-independent event. Taken together, these data indicate that GNG12-AS1 has a dual function in controlling cell cycle progression. Transcription of GNG12-AS1 influences the G1 phase of the cell cycle by modulating DIRAS3 expression, whereas the effect on G2/M phase of the cell cycle by GNG12-AS1 is a DIRAS3-independent event, and most likely represents the function of the RNA product. GNG12-AS1 has a trans function regulating cell migration. DIRAS3 overexpression has been linked to cell migration and cell proliferation 32 , so we assayed these cellular phenotypes after GNG12-AS1 knockdown. We found that reducing GNG12-AS1 with either of the siRNAs increased cell migration [fig_ref] Figure 6 |: GNG12-AS1 regulates cell migration independently of DIRAS3 [/fig_ref]. As no difference was seen between the two siRNAs, we conclude that these phenotypic effects are due to the reduction of GNG12-AS1 transcripts and independent of DIRAS3. Indeed, a change in the migration phenotype was also observed in MCF7 cells, where DIRAS3 silencing cannot be reversed by GNG12-AS1 depletion [fig_ref] Figure 6 |: GNG12-AS1 regulates cell migration independently of DIRAS3 [/fig_ref]. We next profiled global transcription changes after knockdown of GNG12-AS1 and found that both siRNAs against this lncRNA induced significant changes in cell adhesion and actin cytoskeleton pathways [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. We focused on mesenchymal epithelial transition factor (MET), a tyrosine kinase receptor for the hepatocyte growth factor, known to regulate cell migration and invasion [bib_ref] An overview of the c-MET signaling pathway, Organ [/bib_ref] [bib_ref] MET signalling: principles and functions in development, organ regeneration and cancer, Trusolino [/bib_ref] and found increased levels of MET [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. In line with MET activation, we found elevated levels of the MET downstream target MAP2K4 (mitogen-activated protein kinase kinase 4) [bib_ref] MET signalling: principles and functions in development, organ regeneration and cancer, Trusolino [/bib_ref]. This suggests that activation of MET signalling and its downstream targets could be responsible for the observed changes in cell migration. Interestingly, reducing specific minor isoforms of GNG12-AS1 containing exons 2 and 3 was sufficient to trigger activation of this signalling pathway. Future research into the secondary structure of the GNG12-AS1 will determine if this function is isoform specific. Finally, we examined whether simultaneously depleting GNG12-AS1 and DIRAS3 would prevent MET activation. Increased MET protein levels were maintained, confirming that GNG12-AS1 regulates MET signalling independently of DIRAS3 [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. Altogether, these data indicate that the GNG12-AS1 transcript inhibits cell migration. In this context, GNG12-AS1 may have a tumoursuppressor function in addition to a role in modulating the expression levels of DIRAS3. # Discussion LncRNAs can function via their RNA product and through the act of their transcription. So far it has been challenging to tease apart these two processes. Here we employed an siRNA-based strategy targeting different regions of GNG12-AS1 and found that siRNA selectively targeting the first exon of GNG12-AS1 resulted in increased expression of DIRAS3 in cis. These findings, together with inverse relationship between GNG12-AS1 and DIRAS3 during the cell cycle and Actinomycin D treatment, indicate that the act of transcribing GNG12-AS1 regulates expression of the active DIRAS3 allele by TI. We subsequently showed that this transcriptional targeting by siRNA to the 5 0 end of GNG12-AS1 is mediated by AGO2, which binds to the GNG12-AS1 TSS as well as to its transcript. Finally, distinct functional consequences were uncovered, depending on whether GNG12-AS1 transcription was disrupted or whether its product was depleted. The first suggestion that siRNA targeting exon 1 of GNG12-AS1 reduced its nascent transcription was with RNA-FISH, where we observed reduced hybridization with intronic probes after treating cells with this siRNA. More concrete biochemical evidence of transcriptional inhibition were demonstrated by decreased Pol II binding at the GNG12-AS1 TSS with ChIP and reduced nascent transcription with nuclear run-on experiments. siRNA targeted to exon 7, while still reducing the overall GNG12-AS1 product, does not upregulate DIRAS3 and does not change Pol II binding, making this siRNA an excellent internal control for our experiments. Consistent with the data on Pol II binding and nascent transcription, we detected a reduction in the active histone modification, H3K4me3 at the GNG12-AS1 TSS when GNG12-AS1 was depleted using siRNA against exon 1, but not with exon 7. Similarly, the histone modification mark H3K36me3, which normally accumulates in gene body where it is associated with transcriptional elongation [bib_ref] Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to..., Krogan [/bib_ref] , was reduced at the 3 0 end of GNG12-AS1. Thus, our findings suggest that siRNA targeting the 5 0 end of GNG12-AS1 led to inhibition of transcriptional initiation and elongation of GNG12-AS1 by directly interfering with Pol II dynamics without inducing changes in DNA methylation and repressive histone marks. To date, all reported cases of exogenous TGS in human cells have used siRNAs against the gene promoter regions or the TSS [bib_ref] Small interfering RNA-induced transcriptional gene silencing in human cells, Morris [/bib_ref] [bib_ref] The antisense strand of small interfering RNAs directs histone methylation and transcriptional..., Weinberg [/bib_ref] [bib_ref] Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells, Napoli [/bib_ref] [bib_ref] Small RNAs targeting transcription start site induce heparanase silencing through interference with..., Jiang [/bib_ref] [bib_ref] Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells, Kim [/bib_ref]. Our study is, therefore, the first to show that targeting the first exon interferes with the transcription of a lncRNA. Indeed, with GNG12-AS1, targeting regions upstream of the TSS had no effect on its transcription, suggesting that transcription needs to be initiated in order to be targeted by siRNA. This may also explain why, although we found a reduction in active histone modifications, we did not observe an increase in repressive chromatin marks. As TGS is achieved through the RNAi machinery 24,25,27,28 , we investigated the engagement of AGO2 in the inhibition of GNG12-AS1 transcription. siRNAs are known to form a complex with AGO2 in the cytoplasm and then shuffle between the cytoplasm and nucleus [bib_ref] Fluorescence correlation spectroscopy and fluorescence crosscorrelation spectroscopy reveal the cytoplasmic origination of..., Ohrt [/bib_ref]. Small-RNA loading is a cytoplasmic process necessary for the nuclear import of AGO2 . It has further been suggested that small RNAs can guide AGO2 to the promoters of ncRNAs and affect their transcription [bib_ref] Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells, Han [/bib_ref] [bib_ref] Antisense transcripts are targets for activating small RNAs, Schwartz [/bib_ref]. Based on these precedents, we would expect that all siRNAs in our experiments would have a similar knockdown efficiency in different cellular compartments, and once the AGO2-siRNA complex was in the nucleus, the functional consequences of the GNG12-AS1 depletion would depend on the region targeted. The presence of AGO2-siRNA complexes in the nucleus and cytoplasm would explain why we could rescue the GNG12-AS1 expression in double knockdown experiments between GNG12-AS1 and AGO2. However, AGO2-siRNA-GNG12-AS1 complexes at the GNG12-AS1 TSS could only be detected in the nucleus when the siRNA was directed to exon 1. As described above, GNG12-AS1 could only be reduced when sequences downstream of its TSS were targeted by siRNA. This finding, together with the RIP results, showing that AGO2 associates with the GNG12-AS1 when cells are treated with siRNA to exon 1, suggest that transcription needs to have been already initiated for siRNA-mediated repression of transcription. Although AGO2-siRNA interactions with chromatin and the lncRNA could be demonstrated by ChIP and RIP, respectively, we do not have direct evidence for siRNA-AGO2 attaching the nascent lncRNA to the chromatin. We propose a model described in [fig_ref] Figure 7 |: Inhibition of transcriptional interference with siRNA [/fig_ref] , where exogenous siRNA complementary to the 5 0 end (exon 1) of GNG12-AS1 recruits AGO2 to GNG12-AS1 and to the chromatin at its TSS. This complex, containing the siRNA and GNG12-AS1 and possibly other proteins, stalls transcription of GNG12-AS1 by Pol II. DIRAS3 expression is enhanced as a consequence of reduced GNG12-AS1 transcription. There are two possible mechanisms whereby an exogenous siRNA in a complex with AGO2 could lead to decreased GNG12-AS1 transcription. AGO2 has slicer activity 47 and this could promote cleavage and degradation of the nascent GNG12-AS1. Alternatively, the AGO2-siRNA complex could present a physical block for Pol II initiation and elongation at the GNG12-AS1 TSS. Indeed, AGO2 has been shown to guide small RNAs to inhibit Pol II recruitment and elongation in human cells [bib_ref] Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells, Napoli [/bib_ref] [bib_ref] Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene..., Janowski [/bib_ref] independently of its slicer activity [bib_ref] Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells, Napoli [/bib_ref] been reported to form a complex with different chromatinmodifying proteins to mediate gene silencing [bib_ref] AGO2 and SETDB1 cooperate in promotertargeted transcriptional silencing of the androgen receptor..., Cho [/bib_ref]. It is therefore possible that AGO2-siRNA-GNG12-AS1 complexes at the TSS may include further proteins that influence Pol II kinetics. Our model does not distinguish between these different but not necessarily mutually exclusive mechanisms. Silencing of imprinted genes is frequently achieved by TI of an antisense lncRNA [bib_ref] Airn transcriptional overlap, but not its lncRNA products, induces imprinted Igf2r silencing, Latos [/bib_ref] [bib_ref] Regulation of imprinted expression by macro noncoding RNAs, Latos [/bib_ref]. In these cases, the lncRNA is reciprocally imprinted relative to its target gene and is expressed on the opposite allele. Unlike, the other imprinted genes, we found that it was the already actively expressing DIRAS3 allele being upregulated after GNG12-AS1 knockdown, indicating that GNG12-AS1 has a role in modulating DIRAS3 expression in cis, rather than maintaining its imprinting. A reduction of Pol II and H3K4me3 at the TSS of GNG12-AS1 was associated with a concomitant increase at the DIRAS3 TSS. These shifts reflect the change in dynamics between these two genes as transcription inhibition by the GNG12-AS1 is eased and DIRAS3 transcription is augmented. TI between GNG12-AS1 and DIRAS3 could only be consistently inhibited when siRNAs were target within the first exon of GNG12-AS1. Targeting the adjacent intron did not lead to DIRAS3 upregulation in both of the cell lines tested. As GNG12-AS1 was efficiently reduced, we presume the nascent transcript was targeted, but GNG12-AS1 may also be present as mature unspliced transcripts that can be targeted posttranscriptionally. There are several enhancer signature peaks (histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac)) within this intron as annotated in the ENCODE database [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. It is possible that although the nascent RNA can be inhibited by siRNA targeted to this region, enough transcription has already occurred through the region to prevent DIRAS3 upregulation. In a region with multiple enhancers, some are likely to be tissue specific, which could explain why DIRAS3 expression is differentially upregulated in two different cell lines. The presence of enhancer marks at this locus would fit with GNG12-AS1 being an enhancer-like, cis regulator of DIRAS3 transcription [bib_ref] Long non-coding RNAs and enhancers, Orom [/bib_ref]. The location of these enhancers and their potential specificity for DIRAS3 might explain why GNG12 is not affected by GNG12-AS1 depletion. Another possibility is that GNG12-AS1 expression is limited by GNG12 transcription and its further reduction has no impact on GNG12 expression. An alternative strategy to siRNA for analysing the cis functions of GNG12-AS1 would have been to use CRISPRi, a new technology involving small guide RNAs to recruit a catalytically inactive Cas9 (dCas9) fused to the KRAB transcriptional repressor domain to specific loci 52 . Similar to RNAi, CRISPRi does not affect the underlying gene sequence and was shown to induce transcriptional repression of lncRNAs 52 . However, KRABmediated silencing can spread to adjacent genes 53 , which would confound the detection of genes influenced by TI by a lncRNA. Transcription of lncRNA provides an important mechanism for fine-tuning the expression of genes in cis 54 . There is evidence for lncRNAs having a threshold-depended role in moderating gene regulation rather than an activating or repressive role [bib_ref] linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis, Maamar [/bib_ref]. For example, MALAT1, a lncRNA with several roles in gene regulation, has subtle effects on the expression of its neighbouring genes [bib_ref] The lncRNA Malat1 is dispensable for mouse development but its transcription plays..., Zhang [/bib_ref]. A fine-tuning mechanism could explain why small changes in gene expression observed after depletion of some lncRNAs could be relevant upon specific stress or environmental conditions. Imprinted genes are exquisitely regulated for gene dosage. It is likely that in vivo, the cis regulatory function of GNG12-AS1 in fine-tuning DIRAS3 expression is limited to specific developmental stages or environmental conditions as shown for HOTTIP lncRNA 56 , which has similarly low abundance and expression levels. GNG12-AS1 is not imprinted in non-cancer cell lines, as shown previously by pyrosequencing SNP analysis 31 , but our RNA-FISH results indicate that in a substantial proportion of HB2 cells it may be randomly monoallelic. A growing number of autosomal genes are being shown to be randomly monoallelic at single-cell level [bib_ref] Random monoallelic expression: regulating gene expression one allele at a time, Eckersley-Maslin [/bib_ref]. If 50% of cells with GNG12-AS1 expression are expressing transcripts from the paternal allele, these would be repressing DIRAS3 in cis by TI, whereas on the other 50% of cells where GNG12-AS1 expression is maternal, DIRAS3 would already be silent on this allele. This would be consistent with the suggestion that random monoallelic expression refines gene expression levels in a population of cells [bib_ref] Developmental dynamics and disease potential of random monoallelic gene expression, Gendrel [/bib_ref] [bib_ref] Expression of the tumor suppressor ARHI inhibits the growth of pancreatic cancer..., Lu [/bib_ref]. In addition to GNG12-AS1 having a cis function in moderating DIRAS3 transcription, GNG12-AS1 also regulates cell cycle progression. Transcriptional upregulation of DIRAS3 induced by GNG12-AS1 depletion led to G1 delay associated with the downregulation of cyclin E1. As the G1 delay and cyclin E1 levels were rescued by simultaneous depletion of exon 1 siRNA of GNG12-AS1 and DIRAS3, these data suggest that TI between GNG12-AS1 and DIRAS3 contributes to the regulation of G1 phase of the cell cycle. DIRAS3 was shown to be involved in cell cycle progression [bib_ref] Effects of ARHI on cell cycle progression and apoptosis levels of breast..., Li [/bib_ref] [bib_ref] Expression of the tumor suppressor ARHI inhibits the growth of pancreatic cancer..., Lu [/bib_ref]. The upregulation of DIRAS3 by inhibiting GNG12-AS1 transcription in our study does not have the same effect on cyclin genes as previously reported, where DIRAS3 was ectopically overexpressed 40,59,60 . These differences could be due to the modest upregulation of DIRAS3 induced by transient repression of GNG12-AS1 with siRNA, whereas the latter study ectopically expressed DIRAS3. Nevertheless, our results are consistent with a role for DIRAS3 in cell cycle progression through G1 phase [bib_ref] Effects of ARHI on cell cycle progression and apoptosis levels of breast..., Li [/bib_ref] [bib_ref] Expression of the tumor suppressor ARHI inhibits the growth of pancreatic cancer..., Lu [/bib_ref]. More importantly, these results indicate that disruption of TI between GNG12-AS1 and DIRAS3 can have effects on the cell cycle. RNA-FISH analysis indicated that, in addition to co-localization between intronic and exonic probes, we also found cells in which the signals were separated, suggesting that there may be additional functions for GNG12-AS1. Our cell cycle analysis revealed that GNG12-AS1 depletion with siRNAs to 5 0 end and 3 0 end resulted in a G2/M delay. This effect is most likely due to a DIRAS3-independent function of GNG12-AS1 RNA product. This is further supported by our microarray experiments where both siRNAs induced changes in genes involved in cell cycle progression. In addition, we found that reducing the levels of GNG12-AS1 transcripts whether by siRNAs directed to exon 1 or various other exons of GNG12-AS1 resulted in increased cell migration. This phenotype was observed upon GNG12-AS1 knockdown even in cells where DIRAS3 expression was absent. Expression profiling of GNG12-AS1-depleted cells showed an enrichment for genes involved in cell adhesion, migration and actin cytoskeleton pathways. We were able to validate the upregulation of MET signalling pathway after siRNA-mediated reduction of GNG12-AS1. MET controls many biological functions such as cell migration, invasion and motility, and its aberrant activation can lead to cancer [bib_ref] An overview of the c-MET signaling pathway, Organ [/bib_ref] [bib_ref] MET signalling: principles and functions in development, organ regeneration and cancer, Trusolino [/bib_ref] [bib_ref] Targeting MET in cancer: rationale and progress, Gherardi [/bib_ref]. We have previously shown that GNG12-AS1 is downregulated in breast cancer tumours together with DIRAS3 (ref. [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. Thus, it is possible that GNG12-AS1 extends the tumoursuppressor function of this locus by regulating oncogenes such as MET. In keeping with this, MET was recently shown to be regulated by MEG3 lncRNA in pancreatic tumours [bib_ref] Epigenetic regulation of the lncRNA MEG3 and its target c-MET in pancreatic..., Modali [/bib_ref]. Thus, MET could be a common target of lncRNAs involved in the regulation of cellular processes important for tumour growth. Further experiments are necessary to investigate whether the GNG12-AS1 transcript directly interacts with distant genes, and if so whether this interaction is isoform specific. In summary, GNG12-AS1 is similar to several other lncRNAs that have both cis and trans functions [bib_ref] Androgen-responsive long noncoding RNA CTBP1-AS promotes prostate cancer, Takayama [/bib_ref] [bib_ref] The long non-coding RNA Paupar regulates the expression of both local and..., Vance [/bib_ref]. Although the trans function may be to regulate genes involved in cell cycle and cell migration through direct interaction, the cis function has a role in fine-tuning regulation of DIRAS3 transcription. Altogether, our results demonstrate that an siRNA-based strategy can be employed to successfully separate functions that are due to lncRNA transcription from those of the transcript. # Methods Cell culture and treatments. HB2 (human mammary epithelial cell line) were cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS), 10 ml l À 1 penicillin-streptomycin solution, 5 mg ml À 1 insulin (Sigma) and 1 mg ml À 1 hydrocortisone (Sigma). MCF10A (human breast epithelial cell line) were cultured in MEGM SingleQuots (Lonza). SUM159 (breast cancer cell line) were cultured in Ham's F-12 medium (GIBCO) supplemented with 5% FBS, 5 mg ml À 1 insulin and 1 mg ml À 1 hydrocortisone; MCF7 (breast cancer cell line) and HS27 (human foreskin fibroblasts) were cultured in DMEM with 10% FBS and 10 ml l À 1 penicillin-streptomycin solution. 293FT cells were grown in DMEM (high glucose 1  ; GIBCO, ref. 41965039) supplemented with 10% FBS, 0.1 mM MEM Non-Essential Amino Acids (GIBCO), 6 mM L-glutamine (GIBCO), 1 mM MEM Sodium Pyruvate (GIBCO) and 500 mg ml À 1 Geneticin (GIBCO). All the cells were from American Type Culture Collection and were cultured at 37°C with 5% CO 2 . The cells were treated with Actinomycin-D (Sigma) at a final concentration of 10 mg ml À 1 for 0, 2, 6, 8 and 24 h. Single-molecule RNA FISH. RNA FISH was performed as described [bib_ref] Imaging individual mRNA molecules using multiple singly labeled probes, Raj [/bib_ref]. Cells were grown on 18 mm round #1 cover glass, briefly washed with PBS and fixed with PBS/3.7% formaldehyde at room temperature for 10 min. Following fixation, cells were washed two times with PBS. The cells were then permeabilized in 70% ethanol for at least 1 h at 4°C. Stored cells were briefly rehydrated with Wash Buffer (2  saline sodium citrate buffer (SSC), 10% formamide) before FISH. The Stellaris FISH Probes (GNG12-AS1 intronic (Q670) and exonic probes (Q570), sequences in were added to the hybridization buffer (2  SSC, 10% formamide, 10% dextran sulfate) at the same time to give a final concentration of 250 nM per probe set. Hybridization was carried out in a humidified chamber at 37°C overnight. The following day, the cells were washed twice with Wash Buffer at 37°C for 30 min each. The second wash contained 4,6diamidino-2-phenylindole for nuclear staining (5 ng ml À 1 ). The cells were then briefly washed with 2  SSC and then mounted in Vectashield (Vector Laboratories, H-1000). Images were captured using Nikon TE-2000 inverted microscope with NIS-elements software using a Plan Apochromat  100 objective and Andor Neo 5.5 sCMOS camera. We acquired 25 optical slices at 0.3 mm intervals. Images were deconvolved with Huygens Professional and projected in two dimensions using Volocity Image Software Analysis. Intronic signals were scored to determine the percentage of cells with mono-or biallelic expression. Exonic signals were then scored on the same cells. To score whether intronic and exonic signals co-localize, we selected cells in which both signals were present. Quantitative real-time PCR. RNA (1 mg) was extracted with the RNeasy Kit (QIAGEN) and treated with DNase I (QIAGEN) following the manufacturer's instructions. QuantiTect Reverse Transcription Kit (QIAGEN) was used for cDNA synthesis including an additional step to eliminate genomic DNA contamination. Quantitative real-time PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) with Fast SYBR Green Master Mix (Life Technologies). Thermocycling parameters: 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. Two reference genes (GAPDH and RPS18) were selected. Our microarray data show no significant variation in GAPDH and RPS18 expression in our siRNA experiments in HB2, HS27 and SUM159 cells. Expression levels of GNG12-AS1, DIRAS3, GNG12 and WLS in non-cancer and cancer cell lines are normalized relative to the geometric mean of GAPDH and RPS18 [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. The expression levels using either GAPDH [fig_ref] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3 [/fig_ref] [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref] or the average of GAPDH and RPS18 [fig_ref] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1 [/fig_ref] across siRNA conditions were consistent and reproducible. Thus, we used only GAPDH normalization for further experiments. Exon 7-8 of GNG12-AS1 was used to quantify its expression, if not stated otherwise. To calculate the GNG12-AS1 copy number, standard curve of Ct values was performed by qRT-PCR using dilution series of known concentration of GNG12-AS1 DNA template (variant 1 that contains exon . cDNA was made from RNA extracted from known number of different cell lines. The Ct values were fitted on the standard curve and the number of GNG12-AS1 molecules per cell was calculated. The final value was multiplied by 2, to account for the fact that cDNA is single stranded and DNA templates used to make standard curve is double stranded. The sequences for expression primers are listed in Supplementary siRNA-mediated depletion of GNG12-AS1. RNA and proteins were fractionated as described previously [bib_ref] Analysis of nuclear RNA interference in human cells by subcellular fractionation and..., Gagnon [/bib_ref]. The cells were either untreated (for immunoblot analysis) or transfected with control and GNG12-AS1 siRNAs (for RNA analysis). RNA was isolated from cytoplasmic, nucleoplasmic and chromatin fractions by TRIZOL extraction (Life Technologies) and used for qRT-PCR. Data were normalized to the geometric mean of GAPDH and b-actin levels in each cellular compartment as no normalization controls is equal in all three compartments (for details see ref. [bib_ref] Analysis of nuclear RNA interference in human cells by subcellular fractionation and..., Gagnon [/bib_ref]. MALAT1 and RPS18 were used as positive controls for chromatin and cytoplasmic fractions, respectively. Primers used for this assay are listed in [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Nuclear run-on assay. Nuclear run-on assay was performed as described previously [bib_ref] A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells, Johnsson [/bib_ref]. Cells were grown in 10 cm dishes, trypsinized and centrifuged, and the pellets were washed with 1.5 ml of NP-40 lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.5% NP-40), incubated on ice for 5 min and centrifuged at 1,500g for 5 min. Pellets were then washed again with 1.5 ml NP-40 lysis buffer. The nuclei pellets were resuspended in 70 ml glycerol storage buffer (50 mM Tris-HCl, pH 8, 0.1 mM EDTA, 5 mM MgCl 2 , 40% glycerol) and flash frozen in liquid nitrogen. 60 ml were transferred to fresh nuclease-free tubes and equal amount of the 2  run-on transcription buffer (20 mM Tris-HCl, pH8; 5 mM MgCl 2 , 300 mM KCl, 4 mM dithiothreitol) was added together with 2 mM ATP, CTP, GTP (GE Healthcare) and 1 mM Biotin-16-UTP (Epicentre). The reaction was incubated at 30°C for 45 min. In vitro transcription was stopped by adding TURBO DNase (Ambion) at 37°C for 30 min. Then 100 ml of nuclei lysis buffer (50 mM Tris-HCl, pH 7.5; 5% SDS, 0.125 M EDTA) and Proteinase K (Ambion) were added to the sample and incubated at 37°C for 30 min. RNA extraction was performed using phenol/chloroform/isoamyl alcohol: 25:24:1 (Invitrogen) and chloroform/isoamyl alcohol: 24:1 (Sigma). To precipitate RNA, 3 M sodium acetate pH 5.5 (Life Technologies), GlycoBlue (Life Technologies) and ice-cold 100% ethanol were added. The samples were incubated at À 80°C for 30 min and centrifuged at 12,000g for 15 min. Pellets were washed with ice-cold 70% ethanol and centrifuged at 12,000g for 10 min. The pellets were then resuspended in 30 ml nuclease-free water. RNA samples were then purified on column using RNeasy kit (Qiagen) to remove free Biotin-16-UTPs, according to the manufacturer's instructions. Purified RNA from column was eluted in 30 ml nuclease-free water. Biotinylated RNA from samples was captured using Dynabeads MyOne C1 streptavidin beads (Invitrogen). C1 beads (40 ml per sample) were washed twice with Solution A (100 mM NaOH, 50 mM NaCl) for 3 min and once with Solution B (100 mM NaCl). The C1 beads were then preblocked with BSA (200 mg ml À 1 ) and Yeast tRNA (200 mg ml À 1 ; Invitrogen) mix in Solution B. The beads were NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10406 ARTICLE pre-blocked on a rotor at room temperature for 30 min, collected on magnetic stand and resuspended in Solution B. Beads were aliquoted into nuclease-free tubes and equal amount (20 ml) of RNA was added. Biotinylated RNA was pulled down for 2 h at room temperature. Then the beads were collected on magnetic stand, washed three times with 1  Wash/Binding buffer (2  Wash/Binding buffer: 10 mM Tris-HCl, pH 7.4; 1 mM EDTA, 2 M NaCl). After the final wash, C1 beads with bound RNA were eluted in 30 ml nuclease-free water and used directly for reverse transcription to prepare cDNA. Relative transcription was calculated as: 2^À [MeanCt(gene)-MeanCt(b-actin )]. For GNG12-AS1 knockdown experiments, control transcription was set to 1. b-Actin primer was used for normalization (primers are listed in [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Lentivirus overexpression of GNG12-AS1 in human cells. The different GNG12-AS1 splice variants and negative control vector (scrambled sequence) were first cloned in pJET1.2 plasmid (Fermentas) and then into modified pLenti6.3/TO/ V5-DEST vector (kindly provided by John Rinn, Harvard University) using Gateway cloning strategy. The sequences are listed in Supplementary Data 1. The modified vector does not contain WPRE, the SV40 promoter and the blasticidinresistance gene that could interfere with lncRNA structure and function. Sanger sequencing and restriction digestion using PvuI-HF and BsrGI enzymes confirmed the GNG12-AS1 inserts. Lentiviral transduction of GNG12-AS1 clones was done in 293FT cells using ViraPower (Invitrogen) including, negative and positive control vector containing mCherry. The DNA-Lipofectamine2000 complexes were added to 293FT cells and incubated overnight. Forty-eight and seventy-two hours post transfection, the virus-containing supernatants were harvested, centrifuged at 700g for 5 min at 4°C and filtered with 0.45 mm with additional 0.22 mm filter before being stored at 4°C. To transduce SUM159 cells, 80,000 cells per 12-well plate were seeded in triplicate and transduced with the lentiviruses at a multiplicity of infection of 0.1 together with polybrene (5 mg ml À 1 , Sigma). Transduction efficiency (480%) was verified using positive control vector containing mCherry and measured by FACS Calibur Influx (Beckton Dickinson) using BDFACS Software 1.0.0.650. The FACS data were analysed using FlowJo software (TreeStar Inc). Exon 7-9 of GNG12-AS1 was used to quantify its overexpression (primers are listed in Supplementary [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Microarray analysis. Gene expression analysis was carried out on Illumina Human HT12 version 4 arrays. All data analyses were carried out on R using Bioconductor packages [bib_ref] Bioconductor: open software development for computational biology and bioinformatics, Gentleman [/bib_ref]. Raw intensity data from the array scanner were processed using the BASH [bib_ref] BASH: a tool for managing BeadArray spatial artefacts, Cairns [/bib_ref] and HULK algorithms as implemented in the bead array package [bib_ref] beadarray: R classes and methods for Illumina bead-based data, Dunning [/bib_ref]. Log2 transformation and quantile normalization of the data were performed across all sample groups. Differential expression analysis was carried out using the Limma package [bib_ref] Use of within-array replicate spots for assessing differential expression in microarray experiments, Smyth [/bib_ref]. Differentially expressed genes were selected using a P-value cutoff of o0.05 after application of FDR correction for multiple testing applied globally to correct for multiple contrasts. RNA was extracted from cells (HB2, SUM159) treated with control and GNG12-AS1 siRNAs (exons 1 and 7). The analysis was performed with six biological replicates for each cell line. cDNA synthesis, labelling and array procedure were conducted at the Genomic Facility at the CRUK CI. Pathway analysis was performed using Metacore. MeDIP (methylated DNA immunoprecipitation) qRT-PCR. Genomic DNA was extracted from cells with control and GNG12-AS1 siRNA treated with 100 mg ml À 1 RNaseA (Invitrogen) in 100 ml of Tris-EDTA for 10 min at room temperature and sonicated for 5 min (Diagenode Picoruptor, 30 s ON-30sec OFF) to obtain an average fragments size of 300 bp. DNA was purified using 1.8 volumes AMPURE XP beads (Beckman Coulter) according to the manufacturer's instructions and resuspended in 185 ml of water. The samples were boiled for 10 min and snap frozen on ice for 10 min. Five microlitres were taken as input followed by addition of 20 ml cold 10  meDIP buffer. Meanwhile, 20 ml of Protein G Dynal beads (Invitrogen) were blocked with PBS/0.01% BSA for 30 min at room temperature and washed three times with 1  meDIP buffer. One microlitre of anti-5mC antibody (Diagenode) was conjugated to the washed beads in 200 ml of 1  meDIP buffer for 2 h at room temperature. Antibody-bead conjugates were washed three times with cold 1  meDIP buffer, added to the sample DNA and incubated overnight at 4°C in an overhead rotator. The bound material was washed three times with 200 ml of 1  meDIP buffer at room temperature. Bound and input material were resuspended in 50 ml lysis solution (100 mM Tris-HCl, pH 5.5, 5 mM EDTA, 200 mM NaCl, 0.2% SDS plus 20 mg proteinase K (Invitrogen 25530-049)) and incubated at 60°C for 30 min. DNA was purified using 1.8 volumes AMPURE XP beads (Beckman Coulter) according to the manufacturer's instructions. Validation of Medip was done by qRT-PCR. The list of Medip primers is present in [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Analysis of allelic expression and DNA methylation. Analysis of DNA methylation was by bisulfite conversion with the EZ DNA Methylation-Gold Kit (Zymo Research) and subsequent pyrosequencing as described [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. The list of primers used for this experiment is present in . RNA immunoprecipitation. HB2 cells were transfected with control and GNG12-AS1 siRNAs and RIP was performed from nuclear extracts using mouse IgG (Cell Signaling) and AGO2 (Abcam) antibodies. Quality of cytoplasmic and nuclear extracts was assessed by immunoblot as described previously [bib_ref] Analysis of nuclear RNA interference in human cells by subcellular fractionation and..., Gagnon [/bib_ref]. Eight micrograms of antibody were incubated with 70 ml of Dynabeads Protein G beads (Life Technologies) in total volume of 280 ml for 30 min at room temperature. The antibody-bead complex was incubated with 500 mg of nuclear exactas for 2-3 h at 4°C and then washed three times (5 min each) with equal amount of 1  NLB buffer (20 mM Tris-HCl, pH7.5, 0.15 M NaCl, 3 mM MgCl 2 , 0.3% NP-40, 10% glycerol) supplemented with Complete EDTA free protease inhibitor cocktail (14549800; Roche) and phosphatase inhibitors (1 mM NaF, 1 mM Na 3 VO 4 ) and RNAse OUT (100 U; Life Technologies). The RNA was extracted by addition of 1 ml TRIzol (Life Technologies) to the beads, followed by 1/5 volume of Chloroform (Sigma). 10% of the input lysate was mixed with 1 ml of TRIzol for the total input RNA. After centrifugation at 12,000g for 15 min at 4°C, the aqueous supernatant was transferred to a new tube and RNA was precipitated with 1/10 volume of 3 M sodium acetate pH 5.5 (Life Technologies), 1 volume of isopropanol (Sigma) and 1 ml of GlycoBLue (Life Technologies) at À 80°C for 20 min. After 30 min centrifugation at 12,000g at 4°C, the RNA pellet was washed twice with icecold 70% ethanol. Finally, the pellet was dissolved in 15 ml of RNAse-free water (Life Technologies). We used Power SYBR Green RNA-to-CT 1-Step Kit (Life Technologies) for qRT-PCR. Primers for this assay were GNG12-AS1 (exons 7-8) and U1 and GAPDH (negative controls for AGO2 binding). Primers are listed in Supplementary [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Cell lysis and immunoblot. Total cell lysis and immunoblot were performed as described previously [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref]. Briefly, the cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8, 125 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail (Roche)) and incubated on ice for 25 min. The proteins were denatured, reduced and separated using Nupage Novex 4-12% Bis-Tris Protein Gels (Invitrogen). Secondary antibodies were conjugated with peroxidase, and immunobands were detected with a Supersignal West Dura HRP Detection Kit (Thermo-Scientific). Quantification of immunoblots was done on ImageScanner III (GE HealthCare) using the software package ImageQuant TL 7.0 (GE HealthCare). Uncropped scans of the immunoblots are shown in [fig_ref] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus [/fig_ref]. Antibodies. The list of antibodies is present in . Chromatin immunoprecipitation. ChIP assays were performed as previously described [bib_ref] Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding..., Niemczyk [/bib_ref] with antibodies listed in the . The input and the immunoprecipitated materials were quantified by QubitFluorometer (Life Technologies) with the dsDNA BR Assay Kit (Invitrogen). Thirty micrograms of chromatin and 5 mg of antibody were used for ChIP experiment. The qPCR data were corrected for DNA amount, and enrichment was normalized against the input according to the formula 2-dCt(Ab)-log2(DF)]-[Ct(input)-log2(DF)]. Primer sequences are present in [fig_ref] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of... [/fig_ref]. Cell synchronization. The HB2 cells were plated in six-well plates and 24 h later thymidine (2 mM) was added. The cells were incubated for 18 h, washed three times with PBS and released into thymidine-free medium for 9 h. Thymidine was then added for a further 15 h. The cells were then washed three times with PBS. At this point, the cells in G1/S phase were collected for RNA, FACS and protein analysis (T0) in a serum-rich medium without thymidine. Time points were collected 3 (T3), 8 (T8), 14 (T14), 24 (T24) and 32 (T32) hours later. In the case of serum starvation, HB2 cells were plated in six-well plates and then switched to serum-free medium for 48 h. After starvation (T0), the cells were released into cell cycle by addition of serum and the time points for RNA and protein analysis were collected at 3 (T3), 6 (T6), 10 (T10), 15 (T15) and 34 (T34) hours. Cyclin E1 (G1/S transition marker) levels were used to monitor cell cycle progression by immunoblot. In the case of siRNA treatment, the cells were first transfected with siRNAs and thymidine was added 24 h later for additional 18 h. The procedure was continued as described above. Cell cycle analysis. The HB2 cells were transfected with siRNAs and harvested after 72 h, washed with PBS and fixed with 70% ethanol (the samples were stored for up to 1 week at 4°C). The cells were then washed once with PBS, incubated in PBS containing RNase A (100 mg ml À 1 , Life Tecnologies) for 30 min at 37°C, stained with propidium iodide (20 mg ml À 1 , Life Tecnologies) and incubated on ice in the dark for 30 min. DNA content was analysed by FACS Calibur (Beckton Dickinson) using BD CellQuest Pro Software V6. DNA cell cycle analysis was performed on FlowJo software V9 (TreeStar Inc) to quantify cell cycle distribution. Wound healing assay. Wound healing assay was performed on 24-well (Essen Imagelock) plates in triplicates after treatment of cells with control and siRNAs against GNG12-AS1. Forty-eight hours after siRNA treatment, scratch wounds were induced with 10 ml sterile pipette tip, after which fresh culture medium was added. The IncuCyte 2011A Rev2 software was used to capture and analyse the pictures. The cell migration was followed in time for 24 h using IncuCyte FLR (Essen Bioscience), making measurements in triplicate every 3 h. Statistical analysis. The statistical significance of data was determined by twotailed Student's t-test in all experiments using GraphPad Prism unless indicated otherwise. P-values40.05 were considered statistically not significant. [fig] Figure 1 |: GNG12-AS1 is a stable lncRNA in the nucleus. (a) Schematic representation of the [/fig] [fig] Figure 2 |: Transcriptional interference by GNG12-AS1 regulates DIRAS3. (a) The GNG12-AS1 locus where X indicates the exons targeted by siRNA. (b) Changes in single-molecule RNA FISH signals after treating SUM159 cells with siRNA directed to exon 1 or exon 7 of GNG12-AS1. Changes in nascent transcription after exon 1, but not exon 7 siRNA, of GNG12-AS1 were found using intronic probes (left panel). Both siRNAs decreased the number of exonic FISH signal (right panel). Cells positive for intronic probes include cells with both mono and biallelic signal, and cells positive for exonic probes include cells having one or more dots. The number of cells positive for RNA FISH signal is presented relative to control siRNA (control siRNA ¼ 454 cells; exon 1 siRNA ¼ 429 cells; exon 7 siRNA ¼ 257 cells). (c,d) siRNA-mediated knockdown of exon 1 (5 0 targeting) and exon 7 of GNG12-AS1 (3 0 targeting) in HB2 (mammary epithelial cell line, c) and SUM159 (breast cancer cell line, d). Similar results were obtained in two additional cell lines (SupplementaryFig. 3a,b). DIRAS3 is upregulated only when the 5' end of GNG12-AS1 is targeted by siRNA directed to exon 1. For all the graphs, expression levels of DIRAS3, GNG12, WLS, GNG12-AS1 were measured by qRT-PCR, normalized to GAPDH or to geometric mean of GAPDH and RPS18 (see alsoSupplementary Fig. 3c-f), and are presented relative to control siRNA. Primers spanning exons 7-8 were used for GNG12-AS1 expression. RPS18 was used as a negative control gene whose expression does not change upon siRNA treatment. Error bars, s.e.m. (n ¼ 3 biological replicates). *Po0.05, **Po0.01, ***Po0.001 and ****Po0.0001 by two-tailed Student's t-test. [/fig] [fig] Figure 3 |: Inhibition of Pol II after siRNA targeted to exon 1 of GNG12-AS1. (a) The GNG12-AS1 locus showing the location of primer sets (a-g) used for chromatin immunoprecipitation (ChIP) analysis. GNG12-AS1 TSS (a), region 1.6 kb downstream of GNG12-AS1 TSS (b), exon 2 of DIRAS3 (c), DIRAS3 TSS (d), GNG12-AS1 exon 7 (e), GNG12-AS1 exon 8b (f) and GNG12-AS1 exon 9 (g). The X indicates the exons where siRNAs were targeted. (b) Pol II ChIP analysis in HB2 cells treated with siRNA to exons 1 or 7 and controls. ChIP enrichments are presented as the percentage of protein bound, normalized to input. siRNA targeting exon 1 of GNG12-AS1, but not exon 7, results in reduced Pol II binding at GNG12-AS1 TSS (a). At the same time, increased Pol II binding was observed at the DIRAS3 TSS (d). (c) Nuclear run-on analysis of GNG12-AS1 and DIRAS3 transcription after treatment of HB2 cells (left panel) or HS27 cells (right panel) with control and exon 1 or exon 7 GNG12-AS1 siRNA. siRNA targeting exon 1 results in reduced GNG12-AS1 nascent transcription, which leads to increased DIRAS3 transcription. Data are normalized to b-actin and standardized to control siRNA, which is set up as 1 and shown as dotted line. (d,e) ChIP analysis from HB2 cells treated with siRNA to exons 1 or 7 using H3K4me3 (d) and H3K36me3 (e) antibodies. The precipitated DNA fragments were subjected to qRT-PCR analysis with the same primers as for Pol II ChIP. siRNA targeting exon 1, but not exon 7, of GNG12-AS1 reduced H3K4me3 at GNG12-AS1 TSS (a) and H3K36me3 levels in the body of GNG12-AS1 (e-g). Concomitantly, H3K4me3 and H3K36me3 levels increased at the DIRAS3 (d,c, respectively). Levels of H3K4me3 and H3K36me3 were normalized to histone H3 density. For all the graphs, ChIP enrichments are presented as the percentage of protein bound, normalized to input. Neg ctrl ¼ negative control region. Error bars, s.e.m. (n ¼ 3 biological replicates). *Po0.05, **Po0.01 and ****Po0.0001 by two-tailed Student's t-test. [/fig] [fig] Figure 4 |: AGO2 mediates transcriptional inhibition of GNG12-AS1. (a) HB2 cells were transfected with siRNA to exon 1 or exon 7 of GNG12-AS1 together with control siRNA and siRNA targeting AGO2 (A2) (a) or AGO1 (A1) (Supplementary [/fig] [fig] Figure 5 |: DIRAS3-dependent and independent cell cycle regulation. (a,b) Expression patterns of GNG12-AS1 and DIRAS3 are inversely correlated in synchronized cells. HB2 cells were synchronized by double thymidine block (a) or serum starvation (b) and released at indicated time points. Expression levels were measured by qRT-PCR, normalized to GAPDH and standardized to time point 0 h (set as 1). Error bars, s.e.m. (n ¼ 3 biological replicates). (c,d) Immunoblot of cyclin E1 (G1/S transition marker) was used to monitor cell cycle progression of HB2 cells synchronized with a double thymidine block (c) or serum starvation (d). b-Tubulin was used as a loading control. (e) FACS analysis of asynchronous HB2 cells after depletion of GNG12-AS1, DIRAS3 or simultaneous depletion of GNG12-AS1 and DIRAS3. The histograms are representative images of four biological replicates. (f) Cell cycle distribution of HB2 cells following siRNA conditions as shown in e. Note the restoration of G1 cell number between GNG12-AS1 exon 1 siRNA-treated cells and simultaneous depletion of GNG12-AS1 and DIRAS3 (control siRNA: 34% versus exon 1 siRNA: 29%, *Po0.05 by two-tailed Student's t-test). Increase in G2/M population was observed between control and exon 1 siRNA (control siRNA: 23% versus exon 1 siRNA: 35%, ***Po0.001) and exon 7 siRNA (control siRNA: 23% versus exon 7 siRNA: 30%, *Po0.05 by two-tailed Student's t-test). (g) qRT-PCR of DIRAS3, GNG12-AS1, cyclin E1, cyclin B1 and p21 after GNG12-AS1 and DIRAS3 depletion as shown in e. Expression levels are normalized to GAPDH and presented relative to control siRNA. Error bars, s.e.m. (n ¼ 4 biological replicates). *Po0.05, **Po0.01, ***Po0.001 and ****Po0.0001 by two-tailed Student's t-test. (h) Immunoblot of cyclin E1, cyclin B1 and p21 after GNG12-AS1 and DIRAS3 depletion in HB2 cells. Cyclin E1 was reduced only in exon 1 siRNA-treated cells and partially rescued after simultaneous depletion of GNG12-AS1 and DIRAS3 (compare lane 3 to lane 4). b-Tubulin was used as a loading control. (i) Quantification of immunoblots from (h; n ¼ 2 biological replicates). [/fig] [fig] Figure 6 |: GNG12-AS1 regulates cell migration independently of DIRAS3. (a) Cell migration is increased in HB2, SUM159 and MCF7 cells after GNG12-AS1 depletion. Cells were treated with control, exon 1 and exon 7 GNG12-AS1 siRNAs for 48 h before the wound-healing assay was performed over 24 h time course. Cells depleted with siRNA targeted to exon 1 and exon 7 of GNG12-AS1 have significantly increased cell migration. (b) Validation of gene expression changes in SUM159 cells after depletion of GNG12-AS1 with siRNAs targeting exons 1 and 7. Expression levels of MET and MAP2K4 were measured by qRT-PCR, normalized to GAPDH and compared with control siRNA. The knockdown efficiency of GNG12-AS1 is shown in Supplementary Fig. 2b,f. (c) Immunoblot of MET, MAP2K4 and their active phosphorylated (ph) forms in SUM159 cells after depletion of GNG12-AS1 with siRNAs targeting exons 1, 2, 3 and 7. Asterisk (*) indicates a non-specific band detected with an antibody specific to phosphorylated MET. b-Tubulin was used as a loading control. For all the graphs, error bars indicate s.e.m. (n ¼ 3 biological replicates). *Po0.05, **Po0.01 and ****Po0.0001 by two-tailed Student's t-test. DOI: 10.1038/ncomms10406 | www.nature.com/naturecommunications [/fig] [fig] Figure 7 |: Inhibition of transcriptional interference with siRNA. The top panel depicts how GNG12-AS1 modulates the expression of the active DIRAS3 allele through transcriptional interference. GNG12-AS1 may act as a rheostat for DIRAS3 transcription rate. The lower panel depicts how exogenous siRNA molecules in a complex with AGO2 can bind to both the TSS and GNG12-AS1 to inhibit Pol II, and block further transcription initiation and elongation of GNG12-AS1. As a result of transcriptional silencing of GNG12-AS1, transcriptional interference is reduced leading to increased transcription of DIRAS3. [/fig] [table] Table 2: The amplification efficiency of housekeeping genes was measured with serial dilution of cDNA of each gene from five different cell lines. PCR efficiencies and correlation coefficients (R2) for each primer pair are shown in SupplementaryTable 3. [/table]

Global biogeography of SAR11 marine bacteria ## Figure s3 Shifts in sea surface temperature predicted from IPCC climate models. ## Table s1 Source of sequences making up the SAR11 ITS database. ## Table s2 Metagenome sample sets used in this study. ## Table s3 Environmental data associated with metagenomes used in this study. ## Table s4 Results of DISTLM fitting environmental data to relative abundances of SAR11 phylotypes in 128 surface marine metagenome samples. S1_FB04bi.105 S1_FB04bw.3 S1_FB04bw.2 S1_HTCC1002 S1_A22002 S1_HTCC8022 S1_A22001 S1_A14503 S1_A20213 S1_FB04bw.56 S1_A14505 S1_FB04bw.51 S1_FB04bw.43 S1_FB04bw.86 S1_A14504 S1_A10105 S1_A14501 S1_FB04bi.72 S1_FB04bi.88 S1_EBAC00062A03 S1_HTCC1062 S1_AUG5m24 S1_AUG5m7 S1_HTCC8048 S1a_B0152 S1a_HTCC8049 S1a_APR5m24 S1a_AUG5m28 S1a_DEC5m32 S1a_APR5m197 S1a_DEC5m39 S1a_AUG5m61 S1a_M30058 S1a_HTCC8045 S1a_HTCC8046 S1a_HTCC8038 S1a_M30053 S1a_APR5m155 S1a_DEC5m2 S1a_B0153 S1a_APR5m15 S1a_AUG5m59 S1a_HTCC8041 S2_DEC5m20 S1_FB04bi.105 S1_FB04bi.131 S1_231_Clade1 S1_FB04bi.101 S1_FB04bi.32 S1_FB04bi.55 S1_FB04bi.90 S1_G50120 S1_A10104 S1_G50133 S1_G50092 S1_A14503 S1_A20213 S1_G50163 S1_A22001 S1_A22002 S1_A22003 S1_G50001 S1_G50110 S1_G50117 S1_G50152 S1_HTCC1002 S1_HTCC8010 S1_HTCC8022 S1_FB04bi.125 S1_FB04bi.34 S1_FB04bi.89 S1_FB04bi.94 S1_FB04bi.91 S1_FB04bw.2 S1_FB04bw.44 S1_FB04bi.35 S1_FB04bw.3 S1_FB04bw.59 S1_FB04bw.41 S1_FB04bi.46 S1_FB04bi.115 S1_FB04bi.96 S1_FB04bi.77 S1_FB04bw.67 S1_G50027 S1_A14505 S1_G50059 S1_FB04bi.102 S1_FB04bi.79 S1_FB04bw.27 S1_FB04bw.51 S1_FB04bi.21 S1_G500182 S1_G5000115 S1_G50093 S1_G50209 S1_G500212 S1_G50101 S1_G5000119 S1_G50031 S1_G500012 S1_G500017 S1_G50081 S1_G50186 S1_FB04bi.80 S1_FB04bi.134 S1_G500213 S1_FB04bi.38 S1_FB04bi.71 S1_G50011 S1_G50096 S1_G500072 S1_G5000110 S1_G50062 S1_FB04bi.67 S1_G5000121 S1_G50014 S1_G500013 S1_G500214 S1_FB04bi.52 S1_FB04bi.60 S1_F9P1210_R_F07 S1_G5000112 S1_FB04bi.127 S1_FB04bi.141 S1_FB04bw.46 S1_G50007 S1_FB04bw.56 S1_FB04bw.79 S1_G50216 S1_A10101 S1_FB04bw.89 S1_G50077 S1_G50247 S1_G500014 S1_G50065 S1_FB04bw.40 S1_A14504 S1_FB04bi.122 S1_FB04bw.47 S1_G5000117 S1_G500016 S1_FB04bi.37 S1_G50083 S1_G50148 S1_APR5m175 [formula] S2_M30504 S2_MB13H04 S2_APR5m14 S2_EBAC40E09 S2_APR5m156 S2_OCT5m20 S2_HTCC7215 S2_HTCC7217 S2_HTCC8047 S2_HTCC8051 S2_HTCC7211 S2_HTCC7216 S2_MB11E07 S2_DEC5m17 S2_DEC5m13 S2_M30509 S2_APR5m187 S2_OCT5m99 S2_M70055 S2_AUG5m89 S2_SAR11_HF0010_09O16 S2_M70505 SAR11b_GOS031_JCVI_10954600247 SAR11b_M31504 SAR11b_GOS031_JCVI_10954679347 SAR11b_GOS031_JCVI_10954694980 SAR11b_GOS031_JCVI_10954622925 SAR11b_GOS031_JCVI_10954670523 SAR11b_GOS031_JCVI_10954694685 SAR11b_GOS031_JCVI_10954685770 SAR11b_GOS031_JCVI_10954601602 SAR11b_GOS031_JCVI_10954670403 SAR11b_GOS031_JCVI_10954624274 SAR11b_M30501 SAR11b_GOS031_JCVI_10929551346 SAR11b_GOS031_JCVI_10929594929 SAR11b_GOS031_JCVI_10954601164 SAR11b_GOS031_JCVI_10929600193 SAR11b_M30502 SAR11b_GOS031_JCVI_10954601671 SAR11b_GOS031_JCVI_10954620047 SAR11b_GOS031_JCVI_10954694773 SAR11b_M20251 SAR11b_GOS031_JCVI_10954480052 SAR11b_GOS031_JCVI_10955210660 SAR11b_GOS031_JCVI_10955210661 SAR11b_AUG5m4 SAR11b_DEC5m31 SAR11b_HF0070_01M22 SAR11b_MB12A07 SAR11b_GOS031_JCVI_10954601907 SAR11b_GOS031_JCVI_10954670606 SAR11b_GOS031_JCVI_10954600758 SAR11b_M34504 SAR11b_GOS031_JCVI_10954622305 SAR11b_GOS031_JCVI_10954694153 SAR11b_F9P261000_R_B04 SAR11a_GOS031_JCVI_10954623864 SAR11a_GOS031_JCVI_10954624257 SAR11a_MAY150m16 SAR11a_F9P26500_R_G11 S3Deep_F9P12500_R_H07 S3Deep_GOS031_JCVI_10954695241 S3Deep_M31502 S3Deep_M34503 S3Deep_M74001 S3Deep_M31503 S3Deep_EBAC75002 S3Deep_F9P122000_R_C05 S3Deep_M34501 S3Deep_GOS031_JCVI_10954670077 S3Deep_GOS000c_JCVI_784453_GOS S3Deep_GOS031_JCVI_10954602874 S3Deep_GOS000c_JCVI_718883_GOS S3Deep_EBAC75011E01 S3Deep_M34502 S3Deep_F9P262000_R_E10 S3Deep_HF4000_APKG3108 S3Deep_FEB70m11 S3Deep_GOS000C_JCVI_906829_GOS S3Deep_M305010 S3Deep_FEB70m10 S3Deep_GOS031_JCVI_10929630833 S3Deep_GOS031_JCVI_10954600494 S3Deep_GOS031_JCVI_10954602861 S3Deep_M74003 S3Deep_FEB70m34 S3_DEC5m46 S3_OCT5m28 S3_OCT5m17 S3_AUG5m22 S3_MB11B07 S3_APR5m186 S3_APR5m40 S3_APR5m132 S3_GOS114_JCVI_1103242976413_GS3_GOS000d_JCVI_1535771_GOS000 [/formula] S1_FB04bi.74 S1_FB04bi.43 S1_jul5m5 S1_G2K279 S1_G5000120 S1_G50043 S1_G50142 S1_G50295 S1_FB04bi.100 S1_FB04bi.99 S1_FB04bi.98 S1_FB04bi.18 S1_FB04bi.19 S1_APR14m121 S1_G50056 S1_G50226 S1_G500015 S1_G500073 S1_G500142 S1_G500019 S1_FB04bi.130 S1_G500018 S1_G50079 S1_G50172 S1_A10102 S1_FB04bi.10 S1_G50158 S1_G50121 S1_FB04bi.118 S1_FB04bi.85 S1_FB04bi.40 S1_FB04bi.69 S1_G50036 S1_FB04bi.106 S1_FB04bi.128 S1_G50097 S1_FB04bi.137 S1_FB04bw.86 S1_FB04bi.26 S1_FB04bi.11 S1_FB04bi.54 S1_FB04bw.81 S1_G50070 S1_G50173 S1_A10103 S1_G500074 S1_G50197 S1_G50217 S1_G5000116 S1_G50090 S1_G50204 S1_G50237 S1_G50076 S1_G5000118 S1_FB04bi.66 S1_FB04bw.43 S1_FB04bi.14 S1_FB04bi.112 S1_FB04bi.7 S1_G50052 S1_G500093 S1_FB04bi.111 S1_FB04bi.62 S1_FB04bi.68 S1_G50128 S1_A14502 S1_FB04bw.8 S1_FB04bi.110 S1_FB04bi.28 S1_FB04bi.5 S1_FB04bi.30 S1_FB04bw.68 S1_FB04bw.74 S1_FB04bw.82 S1_FB04bi.86 S1_FB04bi.109 S1_G50106 S1_FB04bi.126 S1_G50200 S1_FB04bi.108 S1_FB04bi.132 S1_A22004 S1_FB04bi.78 S1_FB04bi.92 S1_G50207 S1_G50221 S1_FB04bi.121 S1_FB04bi.50 S1_G2K026 S1_G500042 S1_FB04bi.135 S1_G50219 S1_G5000114 S1_G50119 S1_G50210 S1_G50246 S1_G50203 S1_A22005 S1_FB04bi.42 S1_G50273 S1_A10105 S1_G50170 S1_G50064 S1_G50067 S1_FB04bi.103 S1_G50009 S1_FB04bi.72 S1_FB04bi.88 S1_G5000111 S1_G50143 S1_A14501 S1_G50018 S1_G50055 S1_G50063 S1_G50069 S1_G50222 S1_G50211 S1_G500112 S1_FB04bi.84 S1_G50098 S1_G50223 S1_G500092 S1_G50141 S1_G50213 S1_G50248 S1_G50241 S1_G50208 S1_EBAC00062A03 S1_HTCC1062 S1_FB04bw.64 S1_G500113 S1a_APR5m149 S1a_APR5m15 S1a_APR5m170 S1a_APR5m211 S1a_JUL015mB12 S1a_JUL015mB7 S1a_APR14m83 S1a_APR5m117 S1a_APR5m46 S1a_APR5m215 S1a_APR5m231 S1a_M30059 S1a_APR5m155 S1a_APR5m165 S1a_DEC5m2 S1a_DEC5m28 S1a_M30053 S1a_OCT5m63 S1a_APR5m166 S1a_APR5m181 S1a_APR5m193 S1a_HTCC8038 S1a_B0153 S1a_B0152 S1a_HTCC8049 S1a_APR5m24 S1a_AUG5m28 S1a_APR5m164 S1a_AUG5m59 S1a_FEB70m4 S1a_DEC5m32 S1a_APR14m25 S1a_APR14m26 S1a_HTCC8041 S1a_APR14m126 S1a_APR5m140 S1a_JUL015mB1 S1a_APR5m201 S1a_APR5m171 S1a_HTCC8046 S1a_APR14m57 S1a_M30058 S1a_APR14m71 S1a_APR5m136 S1a_AUG5m61 S1a_APR5m115 S1a_APR5m119 S1a_APR5m197 S1a_APR5m137 S1a_APR5m16 S1a_APR5m223 S1a_HTCC8045 S1a_APR5m118 S1a_F9P410_R_G06 S1a_DEC5m39 S1a_APR5m141 S1a_MB11F01 S1a_APR5m135 S1a_APR5m222 S1a_APR5m125 S1a_G50100 S1a_APR5m138 S1a_APR5m194 S1a_G50245 S1a_OCT5m105 S1a_OCT5m74 S1a_M30057 S1a_G50049 S1a_APR5m127 S1a_F9P1210_R_H07 S1a_G50165 S1_AUG5m24 S1_AUG5m7 S1_G50108 S1_G50194 S1_G500312 S1_G50206 S1_G50231 S1_G2K240 S1_G50050 S1_G5009 S1_G50091 S1_G2K088 S1_G2K155 S1_G50235 S1_G50107 S1_G50026 S1_HTCC8048 S1_G50175 HIMB114_HIMB114_HIMB114 HIMB114_HIMB114_JCVI_READ_1092 0.10 1 [fig_ref] Figure S1: ARB neighbour joining trees of SAR11 16S rRNA gene sequences and internal... [/fig_ref] ARB neighbour joining trees of SAR11 16S rRNA gene sequences and internal transcribed spacer (ITS) regions. Normalised distribution of blast hits from genome-specific genes within metagenomes from polar, temperate and tropical biomes. The normalized distribution was calculated by dividing the number of genome-specific protein hits to each biome by the total number of hits to all biomes. Polar <10ºC (blue), temperate 10-20ºC (green), tropical >20ºC (red). [formula] S2_M300510 S2_M70503 S2_M60053 S2_SAR11_HF0010_09O16 S2_APR5m187 S2_M20254 S2_AUG5m89 S2_saSSBWJ41T S2_M60052 S2_DEC5m13 S2_FEB70m32 S2_AUG5m90 S2_OCT5m79 S2_SAR11_fosmid_01_003783 S2_sa2155148 S2_10mA05_HOT S2_saUEAEP53T S2_M30505 S2_M70505 S2_M60055 S2_M70051 S2_M70054 S2_M30509 S2_DEC5m20 S2_M60054 S2_M30504 S2_MB13H04 S2_saSLABR20T S2_M70053 S2_B0154 S2_saSSBAM52T S2_10mH02_HOT S2_sa2072599 S2_saSZAK279T S2_M70052 S2_M70501 S2_M70055 S2_sa1958467 S2_saUDAVA16T S2_saSSBRW35T S2_saUDAXW09T S2_saUDAE424T S2_saUDAR008T S2_M30503 S2_OCT5m99 S2_saUDARP34T S2_M20253 S2_saSKAO856T S2_M60051 S2_saUDANH83T S2_saSXAB906T S2_saSZAF003T S2_AUG5m12 S2_M70504 S2_saSSBS271T S2_sa2059908 S2_HTCC7211 S2_HTCC7216 S2_MB11E07 S2_DEC5m17 S2_M30508 S2_M30507 S2_OCT5m10 S2_APR14m148 S2_OCT5m94 S2_OCT5m20 S2_OCT5m6 S2_APR14m42 S2_APR14m70 S2_HTCC7217 S2_saUAAJC46T S2_HTCC7215 S2_HTCC8047 S2_HTCC8051 S2_M30052 S2_sa2127406 S2_APR5m156 S2_APR5m14 S2_EBAC40E09 S2_M70502 S2_APR5m233 S2_APR14m114 S2_APR14m37 S2_M20252 S2_F9P12500_R_E09 S2_M31505 SAR11b_GOS031_JCVI_10929551346 SAR11b_GOS031_JCVI_10929594929 SAR11b_FEB70m13 SAR11b_GOS031_JCVI_10954600301 SAR11b_GOS031_JCVI_10954601164 SAR11b_GOS031_JCVI_10954601602 SAR11b_GOS031_JCVI_10954670403 SAR11b_GOS031_JCVI_10954624274 SAR11b_M30501 SAR11b_I3K109 SAR11b_I3K115 SAR11b_I3K214 SAR11b_FEB70m29 SAR11b_GOS031_JCVI_10954670523 SAR11b_GOS031_JCVI_10954622925 SAR11b_GOS031_JCVI_10954694980 SAR11b_GOS031_JCVI_10954695570 SAR11b_GOS031_JCVI_10954601368 SAR11b_M31504 SAR11b_GOS031_JCVI_10954600247 SAR11b_GOS031_JCVI_10954670477 SAR11b_GOS031_JCVI_10954685770 SAR11b_GOS031_JCVI_10954694685 SAR11b_GOS031_JCVI_10929600193 SAR11b_GOS031_JCVI_10954679347 SAR11b_G2K241 SAR11b_GOS031_JCVI_10954601671 SAR11b_G2K111 SAR11b_JCVI_READ_2020118 SAR11b_M30502 SAR11b_G2K162 SAR11b_I3K0262 SAR11b_I3K119 SAR11b_I3K026 SAR11b_I3K0263 SAR11b_I3K0264 SAR11b_I3K212 SAR11b_I3K231 SAR11b_GOS031_JCVI_10954620047 SAR11b_GOS031_JCVI_10954694773 SAR11b_M30056 SAR11b_M30506 SAR11b_M30055 SAR11b_M30054 SAR11b_M20251 SAR11b_10mB11_HOT SAR11b_GOS031_JCVI_10954601907 SAR11b_GOS031_JCVI_10954670606 SAR11b_AUG5m27 SAR11b_AUG5m4 SAR11b_AUG5m16 SAR11b_GOS031_JCVI_10955210660 SAR11b_GOS031_JCVI_10955210661 SAR11b_DEC5m31 SAR11b_HF0070_01M22 SAR11b_MB12A07 SAR11b_JCVI_READ_685147 SAR11b_F9P261000_R_A02 SAR11b_G2K140 SAR11b_F9P261000_R_A11 SAR11b_F9P26500_R_C03 SAR11b_F9P122000_R_A04 SAR11b_F9P122000_R_F07 SAR11b_F9P262000_R_A07 SAR11b_F9P4500_R_G11 SAR11b_F9P261000_R_E10 SAR11b_GOS031_JCVI_10954600758 SAR11b_M34504 SAR11b_G2K013 SAR11b_G2K252 SAR11b_F9P26500_R_D11 SAR11b_F9P261000_R_C12 SAR11b_F9P261000_R_B04 SAR11b_FEB70m31 SAR11b_GOS031_JCVI_10954622305 SAR11b_GOS031_JCVI_10954694153 SAR11b_F9P12500_R_B04 SAR11b_I50139 SAR11b_saUEART52T SAR11b_M31501 SAR11b_M74005 SAR11a_F9P26500_R_G11 SAR11a_G2K110 SAR11a_F9P12500_R_G11 SAR11a_G2K116 SAR11a_G2K236 SAR11a_G2K012 SAR11a_G2K2382 SAR11a_G2K0122 SAR11a_F9P4500_R_B11 SAR11a_G2K244 SAR11a_G50149 SAR11a_F9P122000_R_H11 SAR11a_F9P261000_R_B09 SAR11a_G2K271 SAR11a_G2K015 SAR11a_G2K0152 SAR11a_G2K018 SAR11a_G2K027 SAR11a_F9P4500_R_G07 SAR11a_F9P26500_R_B09 SAR11a_G2K253 SAR11a_G2K024 SAR11a_G2K136 SAR11a_G2K085 SAR11a_G2K091 SAR11a_FEB70m3 SAR11a_GOS031_JCVI_10954623864 SAR11a_MAY150m16 SAR11a_F9P12500_R_C03 SAR11a_GOS031_JCVI_10954624257 NA1_G2K054 NA1_G2K250 NA1_GOS031_JCVI_1095462044059_ NA1_G2K259 NA1_F9P122000_R_D01 SAR11b_GOS031_JCVI_10954480052 NA1_G2K228 NA1_G2K154 NA1_G2K045 NA1_G2K152 NA1_G2K097 NA1_G2K164 NA2_F9P41300_R_H07 NA2_G2K082 NA2_I3K096 NA2_G2K275 NA2_F9P121000_R_C07 NA2_F9P26500_R_D01 NA2_I3K128 NA2_F9P261000_R_A04 NA2_M34505 S3Deep_MAY150m5 S3Deep_MAY150m6 S3Deep_MAY150m9 S3Deep_MAY150m7 S3Deep_MAY150m12 S3Deep_MAY150m3 S3Deep_MAY150m2 S3Deep_MAY150m4 S3Deep_F9P122000_R_B08 S3Deep_F9P261000_R_G05 S3Deep_F9P121000_R_F06 S3Deep_EBAC75002 S3Deep_I3K0242 S3Deep_F9P121000_R_D09 S3Deep_F9P121000_R_D12 S3Deep_F9P12500_R_H10 S3Deep_G50104 S3Deep_F9P262000_R_E05 S3Deep_F9P121000_R_C05 S3Deep_gF9P122000_R_G09 S3Deep_F9P262000_R_G05 S3Deep_F9P26500_R_A10 S3Deep_F9P26500_R_D02 S3Deep_G50085 S3Deep_F9P262000_R_E08 S3Deep_F9P262000_R_G06 S3Deep_G2K033 S3Deep_F9P121000_R_H06 S3Deep_MAY500m14 S3Deep_MAY500m17 S3Deep_MAY500m18 S3Deep_MAY500m7 S3Deep_MAY500m9 S3Deep_MAY500m8 S3Deep_F9P122000_R_B05 S3Deep_F9P261000_R_D04 S3Deep_I3K151 S3Deep_I50117 S3Deep_F9P261000_R_D07 S3Deep_F9P261000_R_B03 S3Deep_F9P26500_R_H09 S3Deep_F9P122000_R_D05 S3Deep_F9P262000_R_G04 S3Deep_F9P122000_R_F06 S3Deep_F9P12500_R_B03 S3Deep_FEB70m15 S3Deep_FEB70m36 S3Deep_I3K131 S3Deep_F9P261000_R_F12 S3Deep_F9P261000_R_G01 S3Deep_F9P26500_R_C11 S3Deep_F9P261000_R_B11 S3Deep_F9P41300_R_D04 S3Deep_F9P261000_R_F03 S3Deep_GOS031_JCVI_10954695241 S3Deep_F9P26500_R_H10 S3Deep_F9P121000_R_B02 S3Deep_I3K111 S3Deep_F9P12500_R_A09 S3Deep_F9P12500_R_H05 S3Deep_F9P4500_R_C02 S3Deep_G50029 S3Deep_G500292 S3Deep_G2K086 S3Deep_F9P26500_R_C06 S3Deep_FEB70m11 S3Deep_F9P41300_R_F07 S3Deep_I3K080 S3Deep_F9P122000_R_F10 S3Deep_F9P122000_R_G10 S3Deep_I3K013 S3Deep_I3K0132 S3Deep_I3K135 S3Deep_G2K238 S3Deep_G2K120 S3Deep_G2K122 S3Deep_G2K258 S3Deep_F9P12500_R_F10 S3Deep_I3K132 S3Deep_I3K011 S3Deep_I3K0112 S3Deep_I3K084 S3Deep_I3K144 S3Deep_F9P122000_R_C05 S3Deep_F9P262000_R_B01 S3Deep_F9P261000_R_H10 S3Deep_F9P261000_R_G09 S3Deep_F9P261000_R_H12 S3Deep_F9P12500_R_H07 S3Deep_GOS031_JCVI_10929596383 S3Deep_F9P12500_R_D09 S3Deep_F9P12500_R_D12 S3Deep_GOS000c_JCVI_718883_GOS S3Deep_F9P121000_R_E08 S3Deep_FEB70M16 S3Deep_F9P122000_R_A11 S3Deep_F9P122000_R_C11 S3Deep_MAY890m16 S3Deep_F9P121000_R_E11 S3Deep_F9P121000_R_H05 S3Deep_M34501 S3Deep_GOS031_JCVI_10954670077 S3Deep_F9P121000_R_G02 S3Deep_F9P122000_R_C07 S3Deep_F9P261000_R_F04 S3Deep_B0155 S3Deep_gF9P262000_R_H06 S3Deep_I3K118 S3Deep_M31503 S3Deep_I3K099 S3Deep_F9P4500_R_B05 S3Deep_FEB70m33 S3Deep_I3K025 S3Deep_I3K0252 S3Deep_F9P261000_R_A08 S3Deep_I50144 S3Deep_GOS000C_JCVI_906829_GOS S3Deep_M305010 S3Deep_M74004 S3Deep_FEB70m10 S3Deep_F9P1210_R_H12 S3Deep_F9P1210_R_A08 S3Deep_F9P41300_R_E04 S3Deep_F9P122000_R_C08 S3Deep_F9P261000_R_E04 S3Deep_F9P262000_R_B06 S3Deep_G2K065 S3Deep_F9P122000_R_F12 S3Deep_F9P12500_R_G03 S3Deep_F9P122000_R_A06 S3Deep_F9P262000_R_A12 S3Deep_G2K053 S3Deep_F9P122000_R_H04 S3Deep_I3K023 S3Deep_I3K0232 S3Deep_F9P122000_R_G07 S3Deep_F9P122000_R_G11 S3Deep_F9P122000_R_G06 S3Deep_G2K016 S3Deep_G2K0162 S3Deep_F9P262000_R_A01 S3Deep_I3K104 S3Deep_G2K276 S3Deep_F9P262000_R_D02 S3Deep_I3K015 S3Deep_I3K0152 S3Deep_F9P122000_R_H06 S3Deep_F9P122000_R_C04 S3Deep_I3K117 S3Deep_I3K0233 S3Deep_I3K113 S3Deep_I3K018 S3Deep_I3K0182 S3Deep_G2K087 S3Deep_I3K090 S3Deep_I3K087 S3Deep_I3K022 S3Deep_I3K0222 S3Deep_F9P41000_R_D11 S3Deep_M31502 S3Deep_F9P12500_R_H12 S3Deep_F9P121000_R_C04 S3Deep_F9P121000_R_C06 S3Deep_F9P26500_R_B02 S3Deep_F9P121000_R_H01 S3Deep_F9P261000_R_G11 S3Deep_F9P262000_R_F01 S3Deep_F9P262000_R_G01 S3Deep_F9P122000_R_F09 S3Deep_F9P262000_R_E10 S3Deep_I3K127 S3Deep_I3K106 S3Deep_F9P12500_R_B01 S3Deep_F9P121000_R_F02 S3Deep_F9P261000_R_F07 S3Deep_F9P261000_R_F06 S3Deep_F9P4500_R_A02 S3Deep_I3K003 S3Deep_I3K0032 S3Deep_F9P121000_R_G07 S3Deep_F9P122000_R_E01 S3Deep_gi S3Deep_F9P262000_R_A02 S3Deep_F9P262000_R_A11 S3Deep_F9P262000_R_F03 S3Deep_I3K027 S3Deep_I3K083 S3Deep_F9P121000_R_D05 S3Deep_F9P262000_R_G10 S3Deep_F9P261000_R_B01 S3Deep_F9P261000_R_H03 S3Deep_F9P262000_R_E01 S3Deep_I3K098 S3Deep_I3K042 S3Deep_I3K112 S3Deep_M74001 S3Deep_F9P121000_R_D02 S3Deep_F9P12500_R_F06 S3Deep_F9P41000_R_A04 S3Deep_F9P122000_R_E08 S3Deep_I3K146 S3Deep_M34503 S3Deep_F9P262000_R_F05 S3Deep_I3K093 S3Deep_I3K100 S3Deep_I3K024 S3Deep_I3K0172 S3Deep_I3K125 S3Deep_I3K069 S3Deep_I3K0173 S3Deep_I3K017 S3Deep_F9P12500_R_G08 S3Deep_EBAC75011E01 S3Deep_M34502 S3Deep_HF4000_APKG3108 S3Deep_I3K041 S3Deep_GOS000c_JCVI_784453_GOS S3Deep_GOS031_JCVI_10954602874 S3Deep_I3K097 S3Deep_F9P26500_R_C07 S3Deep_FEB70m28 S3Deep_HF0770_37D02 S3Deep_FEB70m23 S3Deep_F9P262000_R_C07 S3Deep_G50249 S3Deep_M74003 S3Deep_F9P26500_R_H07 S3Deep_F9P1210_R_D11 S3Deep_GOS031_JCVI_10954600494 S3Deep_GOS031_JCVI_10954602861 S3Deep_F9P1210_R_A07 S3Deep_G50296 S3Deep_FEB70m34 S3Deep_GOS031_JCVI_10929630833 S3Deep_I3K095 S3Deep_I3K108 S3Deep_M74002 S3_APR14m14 S3_APR14m143 S3_MB11B07 S3_APR5m128 S3_OCT5m101 S3_OCT5m5 S3_AUG5m22 S3_APR5m106 S3_APR5m199 S3_APR5m150 S3_DEC5m15 S3_DEC5m46 S3_OCT5m17 S3_OCT5m28 S3_APR5m220 S3_OCT5m93 S3_OCT5m19 S3_APR5m186 S3_APR5m47 S3_OCT5m3 S3_APR5m40 S3_GOS114_JCVI_1108839532501_G S3_OCT5m62 S3_MB11D08 S3_GOS000d_JCVI_1477140_GOS000 S3_JCVI_1476732_GOS000D_S3 S3_APR5m132 S3_GOS114_JCVI_1103242976413_G S3_GOS000d_JCVI_1535771_GOS000 S3_OCT5m2 S3_OCT5m66 S3_OCT5m70 S3_GOS000c_JCVI_1018970_GOS000 S3_JUL015mF3 S3_GOS114_JCVI_1108830213766_G S3_OCT5m12 S3_GOS000d_JCVI_READ_1753682_G S3_GOS000d_JCVI_1645073_GOS000 S3_MB21A02 S3_GOS0014_JCVI_1108830148011_ S3a_A20214 S3a_A20216 S3a_A20211 S3a_A20215 S3a_A20212 [/formula] 1 Shifts in sea surface temperature predicted from IPCC climate models. Changes in the location of the 10ºC and 20ºC sea surface temperature isotherms from 1990 (black) and 2090 (red) calculated as a multi-model mean from 15 IPCC models. [fig] Figure S1: ARB neighbour joining trees of SAR11 16S rRNA gene sequences and internal transcribed spacer (ITS) regions. Separate tree files for this figure are provided: Supplementary__ITS_full_MSB.tree; Supplementary_Figure_S1_16S_MSB.tree; Supplementary_Figure_S1_ITS_concise_MSB.tree Figure S2 Normalised distribution of blast hits from genome-specific genes within metagenomes from polar, temperate and tropical biomes. [/fig] [table] Table S5: Specific genes, paralogous genes, and genes with signatures of positive selection in ACE_P1a.1, ACE_P3.2, and IMCC9063 genomes. [/table] [table] Table S1: Source of sequences making up the SAR11 ITS database.Table S2. Metagenome sample sets used in this study. * sample sets generated during this study. [/table] [table] Table S3: Environmental data associated with metagenomes used in this study.* sample sets generated during this study. a Estimated using expectation maximization algorithm in the Primer V6 + PERMANOVER software(Clarke and Gorley, 2006). [/table] [table] Table S4: Results of DISTLM fitting environmental data to relative abundances of SAR11 phylotypes in 128 surface marine metagenome samples. Marginal tests identify the explanatory power of each variable in isolation. The BEST solution identifies the variables best describing the observed phylotype distribution for any given number of combinations. Analyses were carried out with both the full sample set (top) and also on only the 79 samples for which data on all variables was available. SS = sum of squares; Pseudo-F (multivariate analogue of Fishers F ratio, as the pseudo-F statistic gets larger, the likelihood of the null hypothesis being true [ i.e. no relationship between phylotype distribution and environmental variables], diminishes); Prop. = proportion of variation explained by each variable or combination of variables; P = significance by permutation; No. Var = number of the variable; DWC = depth of the water column. [/table]

Child development: analysis of a new concept1 # Introduction Child Development (CD) is a fundamental part of human development, emphasizing that the brain architecture is shaped in the first years, from the interaction of genetic inheritance and influences of the environment in which the child lives [bib_ref] The Lifelong Effects of Early Childhood Adversity and Toxic Stress, Shonkoff [/bib_ref]. To promote the health of children, it is essential to understand their peculiarities, as well as environmental conditions favorable to their development. The understanding of caregivers about the characteristics and needs of children, as a result of their development process, promotes the integral development, because daily care is the major space for the promotion of CD. A valuable resource to the nurse performs their job facing all aspects of child development is the Systematization of Nursing Care. It proposes the use of the nursing classifications, standardizing the language used in assisting individuals, families and communities in different settings [bib_ref] PRONANDA -Programa de Atualização em Diagnósticos de Enfermagem -Conceitos Básicos. Porto Alegre:..., Peres [/bib_ref]. However, for use the nursing classifications in a plan of quality care in the approach of the CD, it is necessary that they address the entire complexity of this phenomenon. A theoretical study of NANDA-International (NANDA-I) and of the International Classification for Nursing Practice (ICNP ® ), which are the most disseminated classifications, found important limitations about the approach of CD [bib_ref] Child Development in the NANDA-I and International Classification for Nursing Practices Nursing..., Souza [/bib_ref]. NANDA-I aims to conduct the language standardization of nursing diagnoses [bib_ref] Diagnósticos de enfermagem e sua relação com o raciocinio clínico, Herdman [/bib_ref]. ICNP ® intends to be an unifying mark of nursing terminologies, and it was recognized by the World Health Organization (WHO) as a member of the Family of International Classifications. NANDA-I has real and risk diagnostics for CD, but there is no promotion diagnostics; these cover the development and growth in a single diagnosis, although they are separate phenomena, with different features and definitions and subject to various interventions [bib_ref] Child Development in the NANDA-I and International Classification for Nursing Practices Nursing..., Souza [/bib_ref]. In ICNP, there are several focus terms related to the child development phenomenon, but these terms do not explain it. The focus terms growth and development are defined as separate terms, but their descriptions are confusing, mixing the two concepts [bib_ref] Child Development in the NANDA-I and International Classification for Nursing Practices Nursing..., Souza [/bib_ref]. When considering the limitations of the CD approach in the two studied classifications, it is possible to suppose a few reasons why the topic has not yet been better studied in both classifications. One of them is the prioritization of biological aspects in health care, with few instruments and approaches that support promotion actions. In this sense, the development of the child is rarely observed in health care. In addition, CD is a broad and complex process, better clarified in recent decades, including its relationship with the daily care and the influence of the environment on it [bib_ref] EarlyHumanDevelopment -Equityfromthe Start -LatinAmerica, Mustard [/bib_ref]. Thus, the difficulties in having nursing diagnoses related to CD can occur in the absence of an approach that encompasses the complexity of the term and the absence of a concept to support the specificity of nursing in the child health. Therefore, it is essential to perform the analysis of the CD concept, to subsidize the classifications of nursing diagnoses and provide diagnostics that enable the development of care plans aimed to CD. This research aimed to perform the concept analysis of the term child development and analyze the new definition proposed as a product of the concept analysis. # Method The concept analysis aims to clarify, recognize and define concepts that describe nursing phenomena, in order to promote understanding. The clarification of a certain concept contributes to the knowledge construction of the area [bib_ref] Conceitos e Teorias na Enfermagem, Bousso [/bib_ref]. The hybrid model of concept development was used in this research. It considers three interconnected stages for the development of the concept: theoretical phase, field phase and analytical phase. In each one of the stages, the four categories of concept analysis must be composed of: attributes, antecedents, consequences and concept definition. issues of temporality, constituting the cronosystem that suppress the changes throughout life. The context is comprised in a broadened form, composed of: microsystem, which includes the nearest environments in which the child lives; mesosystem, which includes interaction of microsystems in which the child is present; exosystems, the environments in which the child is not inserted, but that affect their development, as the work of parents; and macrosystem, which encompasses social and cultural structures and socioeconomic conditions [bib_ref] The bioecological model of human development, Bronfenbrenner [/bib_ref]. # Results In the theoretical phase, 256 articles that met the For the category concept consequences, we selected articles that addressed evaluations of long term CD or in higher age groups, such as school children and teenagers. There were formed two sets of consequences: those relating to adequate CD and those relating inadequate CD. The interviews adressed the same two sets of consequences and the same topics [fig_ref] Figure 1 -: Concept map with the results of the concept analysis of the term... [/fig_ref]. In the category definition of the concept, initially, we grouped articles that discussed more conceptual factors. However, we observed, both in these and in authors of reference on the subject. [fig_ref] Figure 1 -: Concept map with the results of the concept analysis of the term... [/fig_ref] summarizes the analytical phase of the study, of articulation between the data of the field and theoretical phases. The category antecedents is indicated by the connective "is influenced by"; the category attributes is indicated by the connective "is characterized by"; the category consequences is indicated by the connectives "when adequate leads to" and "when inadequate leads to"; and the category definition is indicated by the connective "is defined as". The achievement of the potential of every child depends on the care responsive to their needs of development". # Discussion The results of the concept analysis showed the incorporation of knowledge consistent with the bioecological theory, both in analyzed studies and in the interviews, as pointed out in this discussion, which was structured according to the four elements of the theory process-person-context-time. In Interaction is crucial to development. When relationships are imbued with affection, they allow the formation of a bond that will continue to exist, even when these individuals are not together, being fundamental to the child in the establishment of relations in other social contexts beyond the familiar environment. Still concerning the process, the characteristics of caregivers, especially their mental health, directly affect their interaction with the child. In this sense, the role of the professional can be a factor of support to assist in improving this relationship. A second element of the bioecological theory is the person, considering his/her biopsychological characteristics and those built when interacting with the environment [bib_ref] The bioecological model of human development, Bronfenbrenner [/bib_ref]. In the bioecological model, the characteristics of the person are both producers and products of development, because they are one of the elements that influence the form, content and direction of the proximal processes. The person is in the center of the ecological system [bib_ref] The bioecological model of human development, Bronfenbrenner [/bib_ref]. For the professionals who participated in this study, it seems that environmental aspects supersede the individual, since they stood out. Intrinsic factors of the child were cited in the field phase as factors influencing CD, but with superficial narratives; in the theoretical phase many studies showed the influence of prematurity and low birth weight, child nutrition, growth and diseases. In relation to the context, the environment in which the child is inserted had highlights in the theoretical and field phases, in line with Bronfenbrenner, who stresses the importance of the environment in the context, dividing it into mesosystem, microsystem, exosystem and macrosystem [bib_ref] The bioecological model of human development, Bronfenbrenner [/bib_ref]. In this study, the factors cited of methods that require several steps of analysis. [fig] Figure 1 -: Concept map with the results of the concept analysis of the term child development, according to the process of each child that aims to insert him/ her in the society where he/she lives. It is expressed by continuity and changes in motor, psychosocial, cognitive and language abilities, with progressively more complex acquisitions in the daily life functions. The prenatal period and the first years of life are the foundation of this process, which arises from the interaction of biopsychological characteristics genetically inherited, and experiences offered by the environment. The experiences are constituted by the care that the child receives and the opportunities that the child has to actively exercise his/hers abilities. The care aimed at the needs of development enables the child to reach full potential at every stage of development, with positive repercussions in adult life" (16) . This definition was submitted to the analysis of a group of experts. The focus group had nine participants, besides the researcher who coordinated the group, and her mentor as an observer. Seven nurses, one doctor and one physical therapist participated. There were people from the states of: São Paulo (five), Minas Gerais (two) and Paraná (two). They concluded their undergraduate courses from six to 30 years, but most of them (seven) had from 5 to 15 years of experience. As to professional qualification, one participant had specialization in the area of Neonatal and Pediatric Intensive Care and Public Health Management; two had specialization and master's degree; one had specialization, master's and doctoral degrees; and five had master's degree. Seven participants had publications in the pediatrics area. The group considered that the definition should be more concise, highlighting child development as fundamental to human development, the active role of children in the development process and care as a key element for the promotion of child development. The considerations on the definition of the concept were compatible with the results of the concept analysis, but we realize that they had not been properly incorporated in the first definition proposal. The definition was rewritten as: "Child development is a fundamental part of human development, an active and unique process for each child, expressed by continuity and changes in motor, psychosocial, cognitive and language abilities, with acquisitions progressively more complex in the functions of daily life and in the exercise of their social role. The prenatal period and the first years of child life are crucial in the development process, which is constituted by the interaction of biopsychological characteristics genetically inherited, and experiences offered by the environment. [/fig]

Permeability Measures Predict Hemorrhagic Transformation after Ischemic Stroke Objective: We sought to examine the diagnostic utility of existing predictors of any hemorrhagic transformation (HT) and compare them with new perfusion imaging permeability measures in ischemic stroke patients receiving alteplase only. Methods: A pixel-based analysis of pretreatment CT perfusion (CTP) was undertaken to define the optimal CTP permeability thresholds to predict the likelihood of HT. We then compared previously proposed predictors of HT using regression analyses and receiver operating characteristic curve analysis to produce an area under the curve (AUC). We compared AUCs using χ 2 analysis. Results: From 5 centers, 1,407 patients were included in this study; of these, 282 had HT. The cohort was split into a derivation cohort (1,025, 70% patients) and a validation cohort (382 patients or 30%). The extraction fraction (E) permeability map at a threshold of 30% relative to contralateral had the highest AUC at predicting any HT (derivation AUC 0.85, 95% confidence interval [CI], 0.79-0.91; validation AUC 0.84, 95% CI 0.77-0.91). The AUC improved when permeability was assessed within the acute perfusion lesion for the E maps at a threshold of 30% (derivation AUC 0.91, 95% CI 0.86-0.95; validation AUC 0.89, 95% CI 0.86-0.95). Previously proposed associations with HT and parenchymal hematoma showed lower AUC values than the permeability measure. Interpretation: In this large multicenter study, we have validated a highly accurate measure of HT prediction. This measure might be useful in clinical practice to predict hemorrhagic transformation in ischemic stroke patients before receiving alteplase alone. # Introduction Symptomatic intracranial hemorrhage or hemorrhagic transformation (HT) occurs spontaneously after ischemic stroke. There are few useful predictive markers in clinical practice to identify which patients are at the highest risk of HT. HT ranges from hemorrhagic infarction (HI), which may be a marker of successful reperfusion with minimal or no clinical impact, 1 to large parenchymal hematoma (PH), which substantially increases the risk of poor outcome, including death. [bib_ref] Symptomatic intracerebral hemorrhage in acute ischemic stroke after thrombolysis with intravenous recombinant..., Yaghi [/bib_ref] Treatment with alteplase is associated with a substantially increased risk of HT from 2.4 to 27%, [bib_ref] ECASS Investigators Thrombolysis with Alteplase 3 to 4.5 hours after acute ischemic..., Hacke [/bib_ref] but this risk is balanced against a higher chance of improved clinical outcomes in appropriately selected patients. [bib_ref] Tissue plasminogen activator for acute ischemic stroke, Tniondasr [/bib_ref] [bib_ref] Hacke W Time to treatment with intravenous alteplase and outcome in stroke:..., Lees [/bib_ref] [bib_ref] Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute..., Hacke [/bib_ref] A means of accurately predicting which patients may or may not be at risk of PH or symptomatic intracranial hemorrhage could have significant clinical implications and better inform the treatment decision-making process. Vascular permeability can be measured with magnetic resonance imaging (MRI) or computed tomography perfusion (CTP) [bib_ref] Perfusion and vascular permeability: basic concepts and measurement in DCE-CT and DCE-MRI, Cuenod [/bib_ref] by measuring the rate of contrast leaving a voxel, with the assumption being that a slower or reduced contrast outflow (compared with inflow) is attributable to elevated blood vessel permeability. Elevated permeability may represent disruption or "leakiness" of the blood-brain barrier (BBB), where contrast becomes trapped in the BBB or leaks into the surrounding tissue. Permeability is quantified by analysis of dynamic contrastenhanced images (unprocessed perfusion imaging maps showing contrast flow) to observe decreases in hydrostatic gradients between blood vessels and brain tissue, which can result in contrast lingering around blood vessels. Permeability measures reflect a complex interaction between several physiological variables that affect contrast leakage, such as edema, mass effect, transmural pressure, interstitial fluid pressure, and abnormal tissue biomechanical properties related to BBB permeability. For example, tissue blood volume is associated with abnormal BBB permeability, and several studies have observed a link between decreased cerebral blood volume (CBV) and increased BBB permeability. This is relevant because CBV has been proposed as a marker of increased hemorrhage risk. Permeability maps generated from magnetic resonance perfusion-weighted imaging (PWI) and computed tomography perfusion (CTP) have previously been proposed as measures to predict HT [bib_ref] Does computed tomography permeability predict hemorrhagic transformation after ischemic stroke?, Yen [/bib_ref] [bib_ref] Wintermark M Pretreatment blood-brain barrier damage and post-treatment intracranial hemorrhage in patients..., Leigh [/bib_ref] owing to the proposed relation between permeability maps and blood vessel integrity. However, the accuracy or clinical usefulness of permeability imaging is not yet clear, and there are multiple permutations of permeability measures. [bib_ref] Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data, Patlak [/bib_ref] However, there is evidence suggesting that BBB permeability [bib_ref] Blood-brain barrier permeability is increased in normal-appearing white matter in patients with..., Topakian [/bib_ref] [bib_ref] Weisskoff RM Estimating kinetic parameters from dynamic contrastenhanced T(1)-weighted MRI of a..., Tofts [/bib_ref] can increase in the first few hours after stroke and that elevated permeability measured on CTP might indicate ischemia-induced vascular damage [bib_ref] Velthuis BK Relation between stroke severity, patient characteristics and CT-perfusion derived blood-brain..., Horsch [/bib_ref] and serve as a marker to predict HT. [bib_ref] Gérardin E Increased blood-brain barrier permeability on perfusion computed tomography predicts hemorrhagic..., Ozkul-Wermester [/bib_ref] Previous studies have identified a plethora of clinical risk factors for HT, including advancing age, stroke severity, pretreatment hypertension, and concurrent use of antithrombotic agents. In addition, other studies have identified imaging and laboratory markers, such as hyperglycemia, thrombocytopenia, extensive early ischemic changes on computed tomography (CT), the hyperdense middle cerebral artery sign, very low cerebral blood flow lesion volume, severely delayed blood flow, and large ischemic core volume, which have been related to HT events. [bib_ref] EPITHET Investigators Postthrombolysis blood pressure elevation is associated with hemorrhagic transformation, Butcher [/bib_ref] [bib_ref] Symptomatic intracerebral hemorrhage following thrombolytic therapy for acute ischemic stroke: a review..., Lansberg [/bib_ref] [bib_ref] the Multicenter rt-PA Stroke Survey Group Markers of increased risk of intracerebral..., Tanne [/bib_ref] [bib_ref] Clinical and imaging predictors of intracerebral haemorrhage in stroke patients treated with..., Derex [/bib_ref] However, apart perhaps from major early ischemic change (which is a poor prognostic marker irrespective of HT), none as yet has translated into clinical practice as a routinely used predictor of hemorrhagic transformation. In the present study, we sought to assess the diagnostic utility and association of permeability measures acquired with acute CTP and to identify the optimal cut points for HT outcome in ischemic stroke patients. Next, we sought to compare previously proposed markers of HT with CTP permeability imaging and to assess the comparative accuracies in a large multicenter dataset. We hypothesised that permeability imaging would be an accurate measure of HT of equal or greater value to other validated markers of HT prediction. # Patients and methods Consecutive acute ischemic stroke patients presenting to hospital within 4.5 hours of symptom onset at 5 centers (John Hunter Hospital, New South Wales, Australia; Box Hill Hospital, Melbourne, Victoria, Australia; Royal Adelaide Hospital, Adelaide, South Australia, Australia; Huashan Hospital, Shanghai, China; and Sunnybrook Health Science Centre, Toronto, Ontario, Canada) between 2011 and 2016 were recruited prospectively for the INternational Stroke Perfusion Imaging REgistry (INSPIRE). As part of the registry, patients underwent baseline multimodal CT imaging with non-contrast CT, CTP, and follow-up imaging with MRI or CT at 24 hours post-stroke. Clinical stroke severity was assessed at the two imaging time points using the National Institutes of Health Stroke Scale (NIHSS). All patients in this study were treated with intravenous thrombolysis according to local guidelines and at the clinical judgement of the treating physician. The modified Rankin scale (mRS) was assessed 90 days after stroke. Thrombectomy cases were excluded from this study. Written informed consent was obtained from all participants, and the INSPIRE study was approved by the local ethics committees. ## Acute multimodal ct protocol Participants recruited at John Hunter Hospital and Royal Adelaide Hospital were scanned using a Toshiba Aquilion 320-slice CT scanner (Toshiba Medical Systems, Tokyo, Japan). A total of 19 acquisitions occurred in 60 seconds. Forty milliliters of contrast agent (Ultravist 370; Bayer HealthCare, Berlin, Germany) was injected at 6ml/s, followed by 30ml of saline. Participants recruited at Box Hill Hospital were scanned using a 64-detector GE lightspeed (GE Healthcare, Waukesha, WI ## Twenty-four hour imaging protocol As close as possible to 24 hours after acute imaging, all patients, regardless of treatment, underwent a stroke MRI protocol on a 1.5 or 3T scanner (Siemens Avanto or Verio). The magnetic resonance (MR) protocol included: diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), MR time-of-flight angiography (MRA), and fluid-attenuated inversion recovery (FLAIR) imaging. For those with a contraindication to MRI, repeat noncontrast CT (NCCT) and CTP was performed using the above protocols. # Imaging analysis All perfusion imaging was post-processed with the commercial software MIStar (Apollo Medical Imaging Technology, Melbourne, Victoria, Australia). Acute perfusion imaging was processed using single value deconvolution with delay and dispersion correction. [bib_ref] Perfusion CT in acute stroke: a comprehensive analysis of infarct and penumbra, Bivard [/bib_ref] Areas of no blood flow, chronic infarction or cerebrospinal fluid regions were masked from the perfusion maps. Pixels with no blood flow were removed by eliminating areas where cerebral blood flow = 0, and cerebrospinal fluid/ventricle and skull pixels were removed using a Hounsfield unit threshold and geometric analysis. Previously validated thresholds were applied in order to measure the volume of the acute perfusion lesion (relative delay time [DT] >3 seconds) and acute ischemic core (relative cerebral blood flow [CBF] <30%). [bib_ref] Defining acute ischemic stroke tissue pathophysiology with whole brain CT perfusion, Bivard [/bib_ref] Penumbral volume was calculated as the volume of the perfusion deficit (DT threshold >3 seconds) minus the volume of the ischemic core (relative CBF threshold <30% within the DT >3 seconds lesion). The volume of severely hypoperfused tissue (Tmax >14 seconds) [bib_ref] EPITHET and DEFUSE Investigators Advanced imaging improves prediction of hemorrhage after stroke..., Campbell [/bib_ref] and very low CBV (defined as <2.5th percentile of brain contralateral to the infarct) [bib_ref] Campbell BCV Prediction of poststroke hemorrhagic transformation using computed tomography perfusion, Yassi [/bib_ref] was also recorded for use in HT prediction models. The 24 hour follow-up MRI and/or CT scans were read and Heidelberg Bleeding Classification subtypes categorised by 2 raters (A.B. and M.P.), with any disagreement resolved by a third reader (C.L.). Raters were blinded to all the other imaging and clinical data. Reviewers were blinded to all other imaging. The presence or absence of post-thrombolytic bleeding was then ascertained on 24 hour imaging. Patients were then divided into a target group (HT evident) and a negative HT group. The raters categorised HT in the HT evident group into eight subtypes based on Heidelberg Bleeding Classification as: (1 a ) for hemorrhagic infarction 1 (HI1), scattered small petechiae, no mass effect; (1 b ) for HI2, confluent petechiae, no mass effect; (1 c ) for parenchymal hemorrhage 1 (PH1), hematoma within infarcted tissue, occupying <30%, no substantive mass effect; (2) for PH2, hematoma occupying ≥30% of the infarcted tissue, with obvious mass effect; (3 a ) for parenchymal hematoma remote from infarcted brain tissue; (3 b ) for intraventricular hemorrhage; (3 c ) for subarachnoid hemorrhage; and (3 d ) for subdural hemorrhage. [bib_ref] The Heidelberg bleeding classification: classification of bleeding events after ischemic stroke and..., Von Kummer [/bib_ref] ## Permeability measures All permeability measures were performed using MIStar, and for each patient three permeability maps were generated. The extraction fraction (E) was calculated as the fraction of contrast agent removed from the tissue contrast enactment curve or input residue function during the first pass. At t 1 , the unextracted tracer exits via outflowing blood, and the detector response registers the fraction of extracted tracer, given by E. After t 1 , the extracted tracer diffuses back into the blood and is cleared by outflowing blood, giving rise to a gradually decreasing parenchymal phase. The parameters that can be obtained directly from fitting experimental curves t 1 are the rate of transfer from intravascular to extravascular compartment (k 21 ) and rate of transfer from extravascular to intravascular compartment (k 12 ). Formally, E can be given by: [formula] E = 1−e − k 21t 1 468 Volume 88, No. 3 [/formula] which is a function of the vascular transit time, t 1 . This expression for E implies that, for two capillaries with the same outflow (extravasation) rate, k 21 , the fraction of tracer extracted in the first pass would be larger for the capillary with the longer transit time. [bib_ref] Vogl TJ Prediction of subsequent hemorrhage in acute ischemic stroke using permeability..., Bisdas [/bib_ref] [bib_ref] Tracer-kinetic models for measuring cerebral blood flow using externally detected radiotracers, Larson [/bib_ref] [bib_ref] Computed tomography assessment of cerebral perfusion using a distributed parameter tracer kinetics..., Bisdas [/bib_ref] The permeability-surface area product (P × S, often written PS) is the flow of molecules through the capillary membranes in a certain volume of tissue (expressed in milliliters per minute per 100ml tissue). PS depends not only on the characteristics of the capillary wall, but also on the contrast agent used. PS represents the total diffusional flux across all capillaries and is measured in milliliters per minute per 100g of tissue. It can be interpreted as follows: the unidirectional flux of solutes from blood plasma to the interstitial space is equivalent to the complete transfer of all the solutes in PS milliliters of blood per minute to the interstitial space. In the normal brain parenchyma, BBB is intact and tightly regulated. PS is normally 0 for large hydrophilic molecules, such as a peripherally injected iodinated contrast agent. PS is sometimes called K trans , but PS corresponds to K trans only in very specific conditions. The assumption that back-flux from extravascular compartment into the intravascular one can be neglected during early times depends on the relative magnitude of blood flow (F) and the capillary PS. Permeability (P) is related to the diffusion coefficient of contrast agent in the pores of the capillary endothelium, which are assumed to be water filled. The diffusion flux of contrast agent across the capillary endothelium is dependent on both the diffusion coefficient and the total surface area of the pores or the PS product. The PS product has the same dimensions as F, and thus the ratio PS/F is dimensionless. PS is related to K as follows: [formula] K = EF If PS/F < 1, then K ≈ PS. [/formula] In normal cerebral vasculature, PS is negligible for all contrast agents presently in use. The relative magnitude of PS and F also determines E. [bib_ref] Mozzarelli A Tools for building a comprehensive modeling system for virtual screening..., Kellogg [/bib_ref] [bib_ref] Quantitative estimation of permeability surface-area product in astroglial brain tumors using perfusion..., Jain [/bib_ref] In summary, E is the ratio of contrast leaving a voxel, as opposed to measures such as CBF or CBV, which measure contrast inflow. The transfer constant, K trans , is a complex combination of tissue blood flow and PS in varying proportions, as follows: [formula] K ep = 1 TTP tissue −TTP plasma À Á K trans = V e × K ep V e = C tissue C plasma [/formula] where C tissue is the concentration of contrast in the tissue, C plasma the concentration of contrast in the plasma, TTP tissue the time to reach the peak intensity value in the tissue, and TTP plasma is the time to reach the peak intensity in the plasma in the time-intensity graph for tissue and plasma, respectively. 12 # Permeability analysis For each of the three "types" of the maps defined above (E, K trans and PS), a voxel-based analysis was performed, initially with a whole-hemisphere region of interest and subsequently with the region of interest constrained within the perfusion lesion to define the "location" class (set as a threshold of DT >3 seconds). This results in six individual classes per patient that combine "type" and "location". For each of these six classes individually, a relative threshold at 5% increments (range 0-100%) was tested, resulting in 20 threshold-specific maps (120 maps overall per patient). Threshold-based analysis: In order to identify the optimal threshold to predict HT, for each of the 120 threshold-specific maps a receiver operating characteristic (ROC) curve analysis was used to test the predictive performance of CTP permeability maps at predicting hemorrhagic outcome for: (1) all HT; (2) only HI; (3) only PH (PH1 and PH2); and (4) remote hemorrhage. Results are presented as an area under the curve (AUC) with 95% confidence intervals (CIs) for the whole ROC curve for a perfusion map at a single threshold. Specificity, sensitivity, positive predictive value, and negative predictive value were calculated for each threshold increment (eg, E, K trans , and PS). The optimal thresholds were determined by the highest AUC. All CTP scans with an acquisition or contrast curve (time from the start of the signal increase attributable to contrast arrival) of <45 seconds were excluded from this study owing to the inability to measure K trans and PS accurately from these scans. # Statistical analysis Statistical analyses were programmed using Stata v.13.0 (StataCorp Ltd, College Station, TX). Descriptive results and quantitative baseline patient characteristics were presented as the mean ± standard deviation (SD) or the median and interquartile range. Student paired t tests or Wilcoxon signed-rank tests were performed for parametric or nonparametric data, respectively. The study cohort was split into derivation and validation cohorts for analysis (70 and 30%, respectively), using stratified sampling to ensure that there were no significant differences between the cohorts in terms of baseline characteristics or the occurrences of HT/PH. ## September 2020 469 An ROC curve analysis was used to estimate the AUC, sensitivity, and specificity of each of the permeability maps as a prognostic tool for hemorrhagic outcome. In the ROC analysis, each measurement threshold is a whole analysis and not a point on an ROC curve. Next, an analysis was undertaken to identify whether there was a relation between the volume of the permeability lesion and the Heidelberg HT grade, using ordinal logistic regression. Additionally, patients were analyzed separately based the location of the hemorrhage (either around the area of follow-up infarction or remote from the initial ischemia on CTP). Lastly, once derived, the optimal permeability measure was again assessed within the validation cohort using ROC AUC analysis. The validation ROC AUC values are compared between the pre-specified threshold values to identify the potentially optimal threshold using χ 2 analysis. Once the optimal cut points to predict HT grade were determined, a further ROC AUC analysis was performed using previously identified markers of HT grade (ischemic core volume, very low CBV, Tmax + 14 second volume, reperfusion, diabetes, and age) and compared with the optimal permeability AUC using χ 2 analysis. # Results Throughout the study period, the INSPIRE registry collected 2,127 patients who underwent acute CTP imaging within 4.5 hours of symptom onset and were treated with intravenous alteplase. From the cohort of 2,127 patients, a total of 317 were excluded from the study analysis: 154 because they had incomplete clinical data, and 93 because the imaging could not be processed owing to excessive motion or other technical errors. A further 203 patients were excluded because their acquisition time was <45 seconds. Of the remaining 1,407 patients, 282 developed HTs (91 HI1/1 a , 107 HI2/1 b , 51 PH1/1 c , and 33 PH2/2, 29 of which were symptomatic). Additionally, of the 282 HTs, 47 were 3 a,b,c remote from the infarct and 235 were involving the infarct. The cohort of 1,407 patients were then split into derivation (1,025 patients, 197 HTs) and validation (382 patients, 85 HTs) cohorts for subsequent analysis. Lastly, of the included patients, 493 had follow-up MRI rather than CT (77 HTs, 26 HI1/1 a , 11 HI2/1 b , 23 PH1/1 c , and 17 PH2/2). The baseline characteristics of patients are listed in [fig_ref] TABLE 1: Patient Clinical and Imaging Characteristics [/fig_ref]. There were significant differences between the baseline NIHSS, baseline perfusion lesion volume, baseline ischemic core volume, and treatment types between patients with and without any HT at 24 hours. There were also significant differences in the 24 hour NIHSS, 3 month mRS, and 24 hour core volume between patients with and without any HT at 24 hours [fig_ref] TABLE 1: Patient Clinical and Imaging Characteristics [/fig_ref]. There were no significant differences in the baseline characteristics between the derivation and validation cohorts (p > 0.05). The baseline demographic information was compared between patients with and without a hemorrhagic transformation at 24 h. There were significant differences between the baseline NIHSS, baseline perfusion lesion volume, baseline ischemic core volume, and treatments. There were also significant differences in the 24 h NIHSS, 3 mo mRS, and 24 h core volume between study groups. All study patients were treated with intravenous alteplase. CBF = cerebral blood flow; CBV = cerebral blood volume; mRS = modified Rankin scale; NIHSS = National Institutes of Health Stroke Scale. September 2020 471 The E permeability map of the whole brain at a threshold of 30% had the highest AUC for predicting any HT (derivation AUC 0.85, 95% CI 0.79-0.91; validation AUC 0.84, 95% CI 0.77-0.91; [fig_ref] FIGURE 1: The likelihood of hemorrhagic transformation with the absolute E lesion volume [/fig_ref]. Additionally, an E permeability map of the whole brain at a threshold of 20% was also a reasonably strong predictor of HT (derivation AUC 0.82, 95% CI 0.76-0.88; validation AUC 0.79). Both the PS and K trans maps of the whole brain were relatively poor predictors of HT overall (PS AUC 0.62, validation AUC 0.64; K trans AUC 0.67, validation AUC 0.63). Importantly, permeability maps from E, K trans , or PS were not able to predict remote hemorrhage outside of the acute perfusion lesion in the ipsilesional or contralesional hemisphere (derivation and validation p > 0.5), and remote hemorrhages were excluded from subsequent analyses. These results were maintained in the cohort of patients with MRI at follow-up (E permeability 30%, AUC 0.83, 95% CI 0.76-0.92; K trans AUC <0.6, p > 0.5; PS AUC <0.6, p > 0.5). There was an improved AUC for predicting HT when permeability was restricted to the perfusion lesion (with a threshold of DT >3 seconds) for the E maps at thresholds of 30% (derivation AUC 0.91, 95% CI 0.86-0.95; validation AUC 0.89, 95% CI 0.86-0.95; [fig_ref] FIGURE 2: The likelihood of hemorrhagic transformation with the absolute E lesion volume restricted... [/fig_ref] and 20% (derivation AUC 0.89, 95% CI 0.83-0.95; validation AUC 0.86). Lastly, E permeability maps at a threshold of 30% within the perfusion lesion were also very accurate at predicting patients with PH1/1 c or PH2/2 (derivation AUC 0.93, 95% CI 0.89-0.96; validation AUC 0.9), whereas the K trans and PS were relatively poor (PS AUC 0.67, validation AUC 0.68; K trans AUC 0.66, validation AUC 0.64). An E map at a threshold of 30% within the perfusion lesion had the highest overall AUC at predicting HT across all tested measures . Again these results were maintained in the cohort of patients with MRI at follow-up (E permeability 30%, AUC 0.89, 95% CI 0.78-0.96; K trans AUC <0.6, p > 0.5; PS AUC <0.6, p > 0.5). There was a significant association between the volume of the E permeability map deficit at a threshold of 30% and the severity of the hemorrhagic outcome, with every 10ml increase resulting in an increased chance of a higher grade of hemorrhagic outcome on an ordinal scale (odds ratio 1.13, 95% CI 1.08-1.43, p < 0.001). Moreover, for every 1% increase in the volume of the E map 30% threshold lesion within the perfusion lesion, there was a 1% increase in the likelihood of a PH1/1 c or PH2/2 (odds ratio, 2.01 95% CI 1.83-4.29, p < 0.001). Neither K trans nor PS volumes predicted PH1/1 c or PH2/2 (p > 0.1). For example, if a patient had an E >30% lesion volume that occupied >50% of the perfusion lesion, the likelihood of PH1/1 c was 40% (sensitivity 0.87, specificity 0.81) and that of PH2/2 5% (sensitivity 0.75, specificity 0.83), whereas if only 70% of the perfusion lesion was occupied, the likelihood of PH1/1 c was 70% (sensitivity 0.84, specificity 0.76) and that of PH2/2 50% (sensitivity 0.78, specificity 0.86). However, this result was not maintained in the MRI- With the increasing ratio of E lesion volume to perfusion lesion volume, there was an increased likelihood of hemorrhage and an increase in the severity of hemorrhage. The overall accuracy of the predictive model was high and more reliable than absolute E lesion volume alone. However, it was unusual for an individual patient to have a whole perfusion lesion permeability E 30% lesion, and the results here represent data from a fitted risk model and represent associations rather than true individual patient risk. ## 472 Volume 88, No. 3 FIGURE 3: An example of the E permeability map output (2nd column) and a threshold E permeable map constrained within the perfusion lesion (3rd column), compared with a CTP CBF map (1st column), and the 24 hour CT/MRI (4th and 5th columns). On the E permeability map, the red represents a very prolonged contrast signal, suggesting high permeability. In the threshold map from the 3rd column, the blue area represents the E permeability >30% measure within the perfusion lesion (DT >3 seconds or Tmax >6 seconds). The ability of the E permeability maps to predict HT was significantly increased when a threshold was set at 30% and the lesion volume limited as a fraction of the perfusion lesion. In the first row, there is a small E permeability lesion, which predicted an HI1. The second row has a larger permeability lesion, but after a threshold was applied the fraction of permeability compared with the perfusion lesion was small, and only HI2 was predicted. Next, in the 3rd row, a denser permeability lesion is present, but again the volume was small and an HT2 was predicted. In the 4th row, there is a large, severe permeability lesion, and the patient went on to have a PH2. In the 5th row, the patient has a large perfusion lesion and large permeability lesion area, which developed into a PH2. The last row is an example of a patient with no permeability lesion despite a large perfusion lesion and no HT. CBF = cerebral blood flow; CT = computed tomography; CTP = CT perfusion; DT = delay time; E, extraction fraction; HI = hemorrhagic infarction; HT = hemorrhagic transformation; MRI = magnetic resonance imaging; PH = parenchymal hematoma. September 2020 473 only cohort (odds ratio 1.95 95%, CI 0.83-4.29, p = 0.427). In comparison to previously reported predictors of HT, permeability derived from an E map at a threshold of 30% (AUC 0.91, R 2 = 0.411, p < 0.001, was a stronger predictor than ischemic core volume (AUC 0.62, R 2 = 0.341, p = 0.034, χ 2 p = 0.181), very low CBV (AUC 0.71, R 2 = 0.249, p = 0.026, χ 2 p = 0.382), and severely delayed perfusion (AUC 0.74, Tmax 14 seconds, R 2 = 0.372, p = 0.011, χ 2 p = 0.071). Next, E permeability maps at a threshold of 30% were better at predicting PH than ischemic core volume (AUC 0.77, R 2 = 0.281, p = 0.048, χ 2 p = 0.249), very low CBV (AUC 0.74, R 2 = 0.187, p = 0.041, χ 2 p = 0.124), reperfusion (AUC 0.75, R 2 = 0.182, p = 0.037, χ 2 p = 0.131), and severely delayed perfusion (AUC 0.75, Tmax 14 seconds R 2 = 0.134, p = 0.037, χ 2 p = 0.091). A combined model with an E map lesion volume at a 30% threshold restricted to within the perfusion lesion and ischemic core volume was also highly accurate at predicting PH (AUC 0.92 R 2 = 0.488, p < 0.001, χ 2 p = 0.291), but not HT (AUC 0.74, R 2 = 0.143, p = 0.029, χ 2 p = 0.491). # Discussion From a large, multicenter, international database, we have shown that permeability maps are a strong imaging predictor of HT, including PH1 and PH2, in ischemic stroke patients treated with intravenous alteplase. Additionally, compared with previously described imaging predictors of hemorrhage, E permeability was significantly more accurate, sensitive, and specific at predicting hemorrhage. Moreover, the extent of the E lesion confined within the perfusion lesion was proportional to the severity of hemorrhage across the range of HI1, HI2, PH1, and PH2. Furthermore, the predictive accuracy was very strong, with AUC > 0.9. Not surprisingly, the E permeability maps could not predict remote hemorrhage and were able to predict hemorrhages only when they originated within the acute perfusion lesion. Previously identified markers of HT have insufficient power to alter a treatment decision-making process and have not been implemented in guidelines as contraindications to thrombolysis. The present study provides data to propose a highly accurate predictor of HT likelihood using the E permeability map at a threshold of 30% within the perfusion deficit. Although the E permeability map and threshold cannot predict remote hemorrhage, remote hemorrhage is much less common and probably reflects disorders of fibrinogen production. There is the potential that this measure could be used to alter treatment decisionmaking and, possibly, the consent process. In patients with a large E lesion, for example if a patient had a 60% risk of a PH, this information can be used to inform patient family discussions around expected outcomes and decisionmaking or guide future clinical trials. Additionally, E permeability measures could be useful in trials of HT or PH prevention to isolate a group of patients at risk and enrich the trial cohort. Study limitations need to be acknowledged. Firstly, although INSPIRE is a large, multisite study, in which the sites are strongly encouraged to enroll consecutive patients, the need for pretreatment multimodal CT and follow-up MR, along with clinical data from several time points, means that, practically, not all thrombolyzed patients at the centers were included. [bib_ref] Lansberg MG A benchmarking tool to evaluate computer tomography perfusion infarct core..., Cereda [/bib_ref] Therefore, despite the known baseline clinical and imaging characteristics, some unknown patient cohort biases might have been present, which we cannot taken into account. Secondly, the assessment of the follow-up hemorrhage might have been affected by the use of CT in a proportion of cases, and even in cases with MRI, not all had susceptibility weighted imaging (SWI); therefore, some smaller hemorrhages might have been missed. If a different postprocessing algorithm is used to derive the E measures, then this method would need to be revalidated in a similar manner to the present study; however, the underlying principles are likely to be the same, but might need different thresholds if processed with different algorithms. Importantly, variations in motion correction might also produce different study results, particularly for longer scans, where motion becomes more likely. Next, CTP acquisitions in the present study were of differing lengths (≥45 seconds in length, but the majority were >60 seconds). This might have led to variability in the permeability measures from different centers, particularly for the measures of K trans and PS. Lastly, we did not include patients undergoing thrombectomy at the time of the analysis because we were underpowered, and replication to adjust the statistical model is required. In conclusion, the present study has identified that permeability measures from CTP can accurately predict HT in ischemic stroke patients treated with intravenous alteplase alone. This technique could potentially be useful in trials seeking to target ischemic stroke patients at high risk of HT and might influence the calculation of thrombolysis risk-benefit ratio in individual patients in clinical practice, especially if validated in a thrombectomy cohort. [fig] FIGURE 1: The likelihood of hemorrhagic transformation with the absolute E lesion volume. With increasing E lesion volume, there was an increased likelihood of hemorrhage and an increase in the severity of hemorrhage. [/fig] [fig] FIGURE 2: The likelihood of hemorrhagic transformation with the absolute E lesion volume restricted to the perfusion lesion (DT3 seconds). [/fig] [table] TABLE 1: Patient Clinical and Imaging Characteristics [/table]

Diet-Quality Scores and Prevalence of Nonalcoholic Fatty Liver Disease: A Population Study Using Proton-Magnetic Resonance Spectroscopy Objective Knowledge of the epidemiology of nonalcoholic fatty liver disease (NAFLD) is incomplete because liver biopsy cannot be performed on the general population to assess disease severity. New non-invasive tests allow accurate and safe assessment in healthy individuals. The aim of this study was to examine the prevalence of NAFLD and advanced fibrosis in the general Hong Kong Chinese population. Methods Subjects were recruited from the community by random selection from the government census database. Liver fat and fibrosis were assessed by protonmagnetic resonance spectroscopy and transient elastography, respectively. Results Overall, 264 of 922 (28.6%) subjects had intrahepatic triglyceride content $5%. Excluding 12 subjects with significant alcohol consumption, the population prevalence of NAFLD was 27.3% (95% CI 24.5% to 30.2%). Each component of the metabolic syndrome increased the risk of fatty liver in a dosedependent manner (prevalence of 4.5% in subjects without any component and 80.0% in those with all five components). 8 (3.7%) patients with fatty liver had liver stiffness $9.6 kPa, a level suggestive of advanced fibrosis. Body mass index and alanine aminotransferase level were independent factors associated with liver stiffness. Together with other clinical prediction scores, the estimated prevalence of advanced fibrosis in patients with fatty liver in the community was <10%. Compared with non-drinkers, modest drinkers (<10 g per day) did not have higher risk of fatty liver after adjustment for demographic and metabolic factors. The liver stiffness was 4.761.9 kPa in modest drinkers and 4.661.7 kPa in non-drinkers (p¼0.54). Conclusion NAFLD is found in over a quarter of the general adult Chinese population, but the proportion of patients with advanced fibrosis is low. Modest alcohol consumption does not increase the risk of fatty liver or liver fibrosis. # Introduction Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver function tests worldwide. [bib_ref] Nonalcoholic fatty liver in Asia: firmly entrenched and rapidly gaining ground, Chitturi [/bib_ref] It may progress to cirrhosis and hepatocellular carcinoma, and is believed to be the leading aetiology for cryptogenic cirrhosis. [bib_ref] Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease, Caldwell [/bib_ref] NAFLD is strongly associated with obesity and the metabolic syndrome. As a result, patients with NAFLD have increased mortality from cardiovascular diseases and malignancy in the long run. At present, knowledge of the epidemiology of NAFLD is incomplete. Histological cohorts represent highly selected patients and cannot reflect the situation in the general population. [bib_ref] Clinical and histological features of non-alcoholic fatty liver disease in Hong Kong..., Wong [/bib_ref] Most population studies used abdominal ultrasonography for ## Significance of this study What is already known about this subject? < Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. Although Asia harbours the highest number of diabetic and obese patients, epidemiological data on NAFLD are scarce. < Few studies have evaluated the severity of NAFLD in the general population. New noninvasive tests such as proton-magnetic resonance spectroscopy and transient elastography allow relatively accurate estimation of hepatic steatosis and fibrosis in the community. What are the new findings? < The population prevalence of NAFLD in Hong Kong Chinese was 27.3%. < All components of the metabolic syndrome had an independent association with fatty liver. < Around 4% of subjects with fatty liver in the community had advanced fibrosis as estimated by transient elastography. < Modest alcohol consumption of <10 g per day did not increase the risk of fatty liver and liver fibrosis. How might it impact on clinical practice in the foreseeable future? < The study provides important epidemiological data on NAFLD in the general population. Clinicians may counsel patients based on the study findings. screening. 12e16 However, ultrasonography is operator dependent and is insensitive to mild hepatic steatosis. [bib_ref] The utility of radiological imaging in nonalcoholic fatty liver disease, Saadeh [/bib_ref] In addition, the disease activity of NAFLD relies on histological assessment and is seldom assessed in population studies. In particular, Asia is expected to host the highest number of patients with diabetes and metabolic syndrome owing to its huge population and lifestyle changes. [bib_ref] Diabetes in Asia: epidemiology, risk factors, and pathophysiology, Chan [/bib_ref] Epidemiological data from this region are nevertheless scarce. In recent years, a number of non-invasive tests for liver diseases have been developed. Proton-magnetic resonance spectroscopy ( 1 H-MRS) quantifies hepatic steatosis by measuring proton signals from the acyl groups of hepatocyte triglyceride stores. [bib_ref] Noninvasive assessment of hepatic lipid composition: advancing understanding and management of fatty..., Johnson [/bib_ref] The measurement is reproducible and correlates well with the degree of hepatic steatosis by histology. 20e22 Transient elastography by Fibroscan (Echosens, Paris, France) estimates liver fibrosis by measuring liver stiffness. The measurement has good correlation with histological liver fibrosis in patients with different liver diseases. Its performance has also been validated in patients with NAFLD, and is not affected by the degree of hepatic steatosis and body adiposity. These new tests make population screening possible. In this study, we aimed to study the prevalence of NAFLD and advanced liver fibrosis in the general Chinese population by 1 H-MRS and transient elastography, respectively. We also aimed to study factors associated with NAFLD. # Methods patients This was a cross-sectional study. Subjects from the general population were randomly selected from the census database of the Hong Kong Government, and were invited by mail and phone calls. [bib_ref] High prevalence of colorectal neoplasm in patients with non-alcoholic steatohepatitis, Wong [/bib_ref] We included subjects aged 18e70 years. Subjects with active malignancy, metallic implants or other contraindications to MRI, positive hepatitis B surface antigen or antibody against hepatitis C virus, secondary causes of fatty liver (eg, consumption of amiodarone and tamoxifen) and decompensated liver disease (defined as bilirubin >50 mmol/l, albumin <35 g/l, platelet count <150310 9 /l, international normalised ratio >1.3, or the presence of ascites or varices) were excluded. The study protocol was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong. All subjects provided informed written consent. ## Clinical assessment At the clinic visit, drug history, alcohol intake, smoking and past medical history were recorded using a standardised questionnaire. Alcohol consumption of 20 g daily (140 g weekly) for men and 10 g daily (70 g weekly) for women was the threshold to define NAFLD. [bib_ref] Guidelines for the assessment and management of non-alcoholic fatty liver disease in..., Farrell [/bib_ref] Anthropometric measurements included body weight, body height and waist circumference. Body mass index (BMI) was calculated as weight (kg) divided by height (m 2 ). Waist circumference was measured at a level midway between the lower rib margin and iliac crest with the tape all around the body in the horizontal position. Blood tests including liver biochemistry, glucose and lipids were performed after fasting for 8 h. Metabolic syndrome was defined according to the ethnicspecific criteria by the International Diabetes Federation, which was modified from the National Cholesterol Education Program, Adult Treatment Panel III Guidelines, as any three of the following: (1) central obesity (waist circumference $90 cm in men and $80 cm in women); (2) triglycerides >1.7 mmol/l; (3) reduced high-density lipoprotein-cholesterol (<1.03 mmol/l in men and <1.29 mmol/l in women); (4) blood pressure $130/ 85 mm Hg; and (5) fasting plasma glucose $5.6 mmol/l; or receiving treatment for the above metabolic abnormalities. [bib_ref] Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes..., Alberti [/bib_ref] The degree of liver fibrosis was estimated by transient elastography and clinical prediction formulae. The aspartate aminotransferase (AST) to alanine aminotransferase (ALT) level was calculated as AST/ALT. The AST to platelet ratio index (APRI) was calculated as AST (/upper limit of normal)/platelet count (310 9 /l)3100. [bib_ref] A simple noninvasive index can predict both significant fibrosis and cirrhosis in..., Wai [/bib_ref] FIB-4 was calculated as age 3 AST (IU/l)/platelet count (310 9 /l)3OALT (U/l). [bib_ref] APRICOT Clinical Investigators. Development of a simple noninvasive index to predict significant..., Sterling [/bib_ref] The NAFLD fibrosis score was calculated as À1.675+0.037 3 age (years) +0.094 3 BMI (kg/m 2 ) +1.133 impaired fasting glucose/ diabetes (yes¼1, no¼0) +0.993 AST/ALT ratioe0.0133 platelet count (310 9 /l)e0.663 albumin (g/dl). The BARD score was the weighted sum of three variables (BMI $28 kg/m 2 ¼1 point, AST/ALT ratio $0.8¼2 points, diabetes¼1 point). [bib_ref] Development and validation of a simple NAFLD clinical scoring system for identifying..., Harrison [/bib_ref] Proton-magnetic resonance spectroscopy 1 H-MRS was performed to measure intrahepatic triglyceride (IHTG) content within 8 weeks from the baseline visit. A whole-body 3.0 T scanner with a single voxel point-resolved spectroscopy sequence and an echo time of 40 ms and repetition time of 5000 ms was used. A survey scan was first performed in the abdominal region to help in positioning a volume measuring 20 (AP) 315 (RL) 340 (FH) mm within the liver. The scanner's built-in body coil was used for both signal transmission and reception. A no-water-suppressed spectrum was acquired using 32 signal averages and the data were exported for offline spectral analysis. Water (4.65 ppm) and lipid (1.3 ppm) peak amplitudes were measured to determine vertebral marrow fat content, which was defined as the relative fat signal amplitude in terms of a percentage of the total signal amplitude (water and fat) and calculated according to the following equation: fat content¼ (I fat /(I fat +I water ))3100, where I fat and I water are the peak amplitudes of fat and water, respectively. Correction for relaxation loss was not applied because of the relatively long repetition time and short echo time. An IHTG content of 5% was used to distinguish between patients with and without fatty liver. [bib_ref] Prevalence of hepatic steatosis in an urban population in the United States:..., Browning [/bib_ref] ## Transient elastography Liver stiffness measurement by transient elastography was performed during the baseline clinic visit according to the instructions and training provided by the manufacturer. Measurements were performed on the right lobe of the liver through intercostal spaces with the subject lying in dorsal decubitus with the right arm in maximal abduction. Ten successful acquisitions were performed on each subject. The success rate was calculated as the ratio of the number of successful acquisitions over the total number of acquisitions. The median value represented the liver elastic modulus. Liver stiffness measurements were considered reliable only if 10 successful acquisitions were obtained, the success rate was >60% and the interquartile range to median ratio of the 10 acquisitions was <0.3. Liver stiffness was expressed in kiloPascal (kPa). Operators must have performed at least 50 procedures before participating in this study, and were blinded to all clinical data and the diagnoses of the patients. The cut-off value of 9.6 kPa was used to estimate the number of subjects with advanced fibrosis. This cutoff value had specificity >90% in a previous validation study using liver histology as the reference standard. [bib_ref] Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty..., Wong [/bib_ref] # Statistical analysis Statistical tests were performed using the Statistical Package for Social Sciences version 16.0. Continuous variables were expressed in mean6SD and compared using the unpaired t test if they were normally distributed. Skewed variables were expressed as median (range) and compared using the ManneWhitney U test. QeQ plots were used to screen for normal distribution. Categorical variables were compared using c 2 test or Fisher exact test as appropriate. Binary logistic regression analysis was performed to identify factors associated with fatty liver. The Spearman correlation test was performed to assess the correlation between clinical factors and liver stiffness in patients with fatty liver. Linear regression analysis was performed to identify independent factors associated with liver stiffness. According to the census in 2006, the Hong Kong population aged $15 years was 5 924 671. A sample size of 900 would determine the prevalence of NAFLD in Hong Kong at a 3% CI and 95% confidence level for a prevalence of 20e50%. Assuming 10% of the subjects would be excluded due to viral hepatitis and other reasons, a total sample size of 1000 subjects was required. # Results From May 2008 to September 2010, invitation letters were sent to 3000 randomly selected Hong Kong residents from the census database. One thousand and sixty-nine subjects responded, with a response rate of 35.6%. After excluding subjects with contraindications to or failed 1 H-MRS examination and those with concurrent viral hepatitis, 922 subjects were included in the final analysis (figure 1). The mean age of the subjects was 48611 years (range 19e72 years), and 533 (57.8%) were women (table 1). A total of 48 (5.2%) and 143 (15.5%) subjects had diabetes and hypertension, respectively, and 209 (22.8%) subjects had BMI $25.0 kg/m 2 . All subjects were ethnic Chinese. ## Prevalence of fatty liver Overall, 264 (28.6%) subjects had fatty liver detected by 1 H-MRS. Only 12 subjects with fatty liver had significant alcohol consumption between 140 and 350 g per week. After excluding these subjects, the population prevalence of NAFLD was 27.3% (95% CI 24.5% to 30.2%). Among subjects with fatty liver, 154 (58.3%) had an IHTG content of 5e10.9%, and 110 (41.7%) had an IHTG content $11.0%, a level suggestive of >33% hepatic steatosis by histology. [bib_ref] Magnetic resonance imaging and spectroscopy accurately estimate the severity of steatosis provided..., Mcpherson [/bib_ref] The prevalence of fatty liver was 36.8% (95% CI 32.0% to 41.6%) in men and 22.7% (95% CI 19.1% to 26.3%) in women. In men, the prevalence of fatty liver peaked at 40 years of age and remained relatively constant up to the seventh decade (figure 2). In contrast, the prevalence of fatty liver remained low at 12e16% in women younger than 50 years, and increased steadily after the menopause. By univariate analysis, older age, male gender, high plasma creatinine, ferritin and haemoglobin levels, and components of the metabolic syndrome were associated with fatty liver [fig_ref] Table 2: Factors associated with fatty liver [/fig_ref]. By multivariate analysis, a high ferritin level and components of the metabolic syndrome remained as independent factors associated with fatty liver, while the association with age and male gender became insignificant and could be largely explained by the differences in metabolic factors. All five components of the metabolic syndrome according to the International Diabetes Federation criteria were independently associated with fatty liver [fig_ref] Table 2: Factors associated with fatty liver [/fig_ref]. Moreover, a doseeresponse relationship was observed. The prevalence of fatty liver was only 4.5% in subjects without any component of the metabolic syndrome but 80.0% in those with all five components (figure 3). The liver stiffness was significantly higher in subjects with fatty liver than in controls (5.161.9 kPa vs 4.461.6 kPa; p<0.001) (table 1). Eight (3.7%) patients with fatty liver and 7 (1.3%) controls had liver stiffness $9.6 kPa, a level suggestive of advanced fibrosis (p¼0.032). Among patients with fatty liver, liver stiffness had a positive correlation with obesity, systolic blood pressure, serum ALT and AST levels, and IHTG content. By multivariate analysis using a linear regression model, BMI (b 0.17; p¼0.008) and ALT level (b 0.24; p¼0.019) remained as independent factors associated with liver stiffness. ## Prevalence of advanced fibrosis Based on the calculation of the 95th percentiles, 90% of subjects without fatty liver or other liver diseases had liver stiffness between 2.8 and 7.4 kPa, which reflected the normal range in the general population. The same normal range was found for both men and women when the analysis was stratified by gender. Other than liver stiffness measurement, estimation of liver fibrosis was also performed using the AST/ALT ratio, APRI, FIB-4 score, NAFLD fibrosis score and BARD score. Based on different scores, the estimated prevalence of advanced fibrosis was between 0 and 12.1% in subjects with fatty liver. When lower cut-off values of the scores were used for screening, 83.7e95.5% of subjects with fatty liver were unlikely to have significant fibrosis. ## Validation of the new alt cut-off values Four hundred and twenty-six subjects had elevated ALT according to the new cut-off values ($30 IU/l in men and $19 IU/l in women), and 496 subjects had normal ALT. Fatty liver was found in 193 (45.3%) subjects with elevated ALT and A doseeresponse relationship was observed between ALT level and the prevalence of fatty liver. Seventy-one of 496 (14.3%) subjects with ALT below the new cut-off values, 165 of 387 (42.6%) subjects with ALT between the new cut-off values and the old local laboratory cut-off value of 58 IU/l, and 28 of 39 (71.8%) subjects with ALT >58 IU/l had fatty liver (p<0.001). The median IHTG content of the three groups of subjects was 1.4% (0e35.1%), 4.1% (0e31.2%) and 11.6% (0.7e44.2%) (p<0.001 by KruskaleWallis test). Among 759 subjects with valid liver stiffness measurements by transient elastography, 347 had elevated ALT and 412 had normal ALT. Nine (2.6%) subjects with elevated ALT and 6 (1.5%) subjects with normal ALT had liver stiffness $9.6 kPa (p¼0.26). The liver stiffness was 4.861.8 kPa in subjects with elevated ALT and 4.561.7 kPa in subjects with normal ALT (p¼0.013). ## Effect of modest alcohol consumption Seven hundred and twenty subjects (78.1%) were non-drinkers, 148 (16.1%) had modest alcohol consumption of <70 g per week, and 54 (5.9%) had alcohol consumption $70 g per week. The amount of alcohol consumption had a weak positive correlation with IHTG content by 1 H-MRS (Spearman correlation coefficient 0.11; p¼0.001) but not liver stiffness (Spearman correlation coefficient 0.003; p¼0.94). Fatty liver was detected in 56 (37.8%) modest drinkers and 190 (26.4%) non-drinkers (p¼0.005). The median IHTG content was 2.7% (range 0.1e44.2%) in modest drinkers and 1.9% (range 0e35.1%) in non-drinkers (p¼0.012). After adjustment for age, gender and metabolic syndrome, modest alcohol consumption was no longer associated with fatty liver (adjusted OR 1.37; 95% CI 0.89 to 2.11; p¼0.15). The proportion of subjects with liver stiffness $9.6 kPa was similar between modest drinkers (3 of 130 (2.3%)) and nondrinkers (10 of 583 (1.7%)) (p¼0.72). The liver stiffness was 4.761.9 kPa in modest drinkers and 4.661.7 kPa in non-drinkers (p¼0.54). # Discussion In this large epidemiological study in the general adult Chinese population, the prevalence of NAFLD is 27.3%. Around 4% of patients with fatty liver have advanced fibrosis. Fatty liver was found in 14.3% of subjects with normal ALT according to the updated stringent cut-off values (<30 IU/l in men and <19 IU/l in women). Modest alcohol consumption increases hepatic fat but does not increase the risk of advanced fibrosis. Compared with previous reports, our study recruited asymptomatic individuals from the general population by using the government census database. The use of state-of-the-art non-invasive tests allowed accurate assessment of hepatic steatosis and fibrosis in a large number of subjects in the community, in which liver biopsy may not be feasible. Previous epidemiological studies reported a prevalence of NAFLD of 20e40% in the USA and Europe. [bib_ref] Prevalence of and risk factors for hepatic steatosis in Northern Italy, Bellentani [/bib_ref] Owing to westernisation of lifestyle, obesity and metabolic syndrome are increasingly prevalent in Asia. A summary of Asia-Pacific studies showed that the prevalence of NAFLD was 16e42%. [bib_ref] How common is non-alcoholic fatty liver disease in the Asia-Pacific region and..., Amarapurkar [/bib_ref] However, many studies from Asia were limited by the lack of a uniform definition of NAFLD and the inclusion of hospital patients instead of community subjects. As such, our current study confirms that the prevalence of NAFLD in the Chinese population is similar to that in western countries. The prevalence of NAFLD is higher in men and postmenopausal women. The underlying cause is not completely understood, but dysregulated sex hormones appear to affect the liver in various ways. For example, NAFLD is prevalent in women with polycystic ovary syndrome. [bib_ref] Nonalcoholic fatty liver disease in women with polycystic ovary syndrome, Cerda [/bib_ref] In patients with viral hepatitis, male gender is associated with disease progression and higher risk of hepatocellular carcinoma. In line with previous histological series, metabolic syndrome has the strongest association with NAFLD. Our current study suggests that all components of metabolic syndrome have independent and important association with NAFLD. It would be important to investigate the relative contribution by different metabolic factors to NAFLD progression in longitudinal studies. [bib_ref] Disease progression of non-alcoholic fatty liver disease: a prospective study with paired..., Wong [/bib_ref] In addition, ferritin is another independent factor associated with fatty liver. While ferritin is an acute phase protein that may be increased in a systemic inflammatory state, the interaction between iron stored and NAFLD has been underscored by various studies. Since ultrasound scan can only detect fatty liver but cannot assess disease severity, current knowledge of the impact of NAFLD is incomplete. Recently, Williams and colleagues reported a study of 328 patients at the Brooke Army Medical Center. [bib_ref] Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely..., Williams [/bib_ref] All patients with fatty liver detected by ultrasound scan were invited to have liver biopsy examination. Overall, 151 (46%) patients had fatty liver. Among 134 patients with fatty liver who underwent liver biopsy, 40 (30%) had non-alcoholic steatohepatitis and 9 (7%) had advanced fibrosis. Since patients in this study were recruited from the clinic, it was not actually a population study. The high prevalence of diabetes and obesity in this cohort also explains the high proportion of patients with NAFLD and steatohepatitis. Nevertheless, this is already one of the best studies addressing the prevalence and severity of NAFLD in a primary care setting. With the use of 1 H-MRS and transient elastography, our current study was able to assess both hepatic steatosis and fibrosis. Based on liver stiffness measurements and confirmed by various clinical prediction models of liver fibrosis, the prevalence of advanced fibrosis in patients with fatty liver is w4% and unlikely to exceed 10%. Thus, at most, 3% of the general population would have advanced fibrosis secondary to fatty liver. Since the population prevalence of NAFLD is high, further studies should be conducted to determine the value of 1 H-MRS screening for at-risk groups such as those with metabolic syndrome. Unlike full MRI, 1 H-MRS can be done within 20 min. With development of new technologies, the test may even be combined with magnetic resonance elastography for liver fibrosis assessment and phosphorus-MRS for liver injury. This may provide clinicians and patients with a comprehensive but non-invasive evaluation of the liver. Recently, it was recognised that old ALT cut-offs tend to be too high because the normal range was derived from healthy volunteers who might harbour fatty liver. A set of updated ALT cut-off values (30 IU/l in men and 19 IU/l in women) was proposed to improve the accuracy in detecting patients with liver diseases. [bib_ref] Updated definitions of healthy ranges for serum alanine aminotransferase levels, Prati [/bib_ref] In fact, patients with high-normal ALT are at increased risk of liver-related mortality in the long run. [bib_ref] Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective..., Kim [/bib_ref] In our study, we confirmed that subjects with normal ALT according to the updated cut-offs were less likely to have fatty liver. However, ALT only had a weak correlation with liver stiffness. In histological series of patients with NAFLD, a normal ALT level could not reliably exclude steatohepatitis and advanced fibrosis. Patients progressing to cirrhosis may even have burnt-out disease and a paradoxical drop in ALT level. [bib_ref] Histological progression of non-alcoholic fatty liver disease in Chinese patients, Hui [/bib_ref] Therefore, while the prevalence of NAFLD is generally lower in people with normal ALT, ALT is a poor marker of disease severity in patients with NAFLD. Modest alcohol consumption protects the cardiovascular system. Its effect on fatty liver is unclear. A posthoc analysis of the Third National Health and Nutrition Examination Survey suggested that modest wine drinkers (<10 g per day) had lower risk of fatty liver. [bib_ref] Modest wine drinking and decreased prevalence of suspected nonalcoholic fatty liver disease, Dunn [/bib_ref] However, that study used elevated ALT to define fatty liver and no imaging studies were performed. In our study, even modest drinkers had a higher prevalence of fatty liver, although the effect appears to be less important after adjustment for other metabolic factors. Moreover, our data clearly demonstrate that modest drinking does not increase the risk of advanced fibrosis. Our study has a few limitations. First, all subjects were ethnic Chinese with a low prevalence of excessive alcohol consumption. Our findings cannot reflect the situation in different countries. However, the case recruitment from a government census database and the use of 1 H-MRS and transient elastography made this one of the few studies in the literature that provide comprehensive epidemiological data on NAFLD. Secondly, while transient elastography has excellent negative predictive value in excluding advanced fibrosis in patients with NAFLD, the positive predictive value in diagnosing the condition is imperfect. [bib_ref] Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty..., Wong [/bib_ref] Therefore, we minimised the bias by supplementing the data with five other clinical prediction scores. The results consistently confirmed a low rate of advanced fibrosis in patients with NAFLD in the general population. Thirdly, IHTG was measured once and we cannot exclude the possibility that the value may change with time. However, a previous study in college students showed that while a shortterm increase in food intake might change the metabolic profile and ALT levels, the effect on IHTG was modest. [bib_ref] Fast-food-based hyper-alimentation can induce rapid and profound elevation of serum alanine aminotransferase..., Kechagias [/bib_ref] The small variability in IHTG would be unlikely to affect the major observations in this study. Finally, we did not exclude rarer liver diseases such as autoimmune hepatitis, primary biliary cirrhosis and metabolic liver diseases. However, the population prevalence of these diseases is low and would be unlikely to affect the results significantly. Similarly, the prevalence of non-alcoholic steatohepatitis is unknown as liver biopsy was not performed. However, recent data suggest that liver fibrosis is more important than other histological features of steatohepatitis in determining the long-term liver-related mortality of patients with NAFLD. [bib_ref] Pathologic criteria for nonalcoholic steatohepatitis: Interprotocol agreement and ability to predict liver-related..., Younossi [/bib_ref] In conclusion, NAFLD is found in over a quarter of the general adult Chinese population, but the proportion of patients with advanced fibrosis is low. Modest alcohol consumption increases the risk of fatty liver but does not affect liver fibrosis. [fig] Figure 1: Study recruitment and participant flow. [/fig] [fig] Figure 3: Prevalence of fatty liver in subjects harbouring different numbers of components of the metabolic syndrome according to the International Diabetes Federation criteria. [/fig] [table] Table 1: Clinical characteristics of subjects with and without fatty liver [/table] [table] Table 2: Factors associated with fatty liver [/table]

The grain quality of wheat wild relatives in the evolutionary context Key message We evaluated the potential of wheat wild relatives for the improvement in grain quality characteristics including micronutrients (Fe, Zn) and gluten and identified diploid wheats and the timopheevii lineage as the most promising resources. Abstract Domestication enabled the advancement of civilization through modification of plants according to human requirements. Continuous selection and cultivation of domesticated plants induced genetic bottlenecks. However, ancient diversity has been conserved in crop wild relatives. Wheat (Triticum aestivum L.; Triticum durum Desf.) is one of the most important staple foods and was among the first domesticated crop species. Its evolutionary diversity includes diploid, tetraploid and hexaploid species from the Triticum and Aegilops taxa and different genomes, generating an AA, BBAA/GGAA and BBAADD/GGAAA m A m genepool, respectively. Breeding and improvement in wheat altered its grain quality. In this review, we identified evolutionary patterns and the potential of wheat wild relatives for quality improvement regarding the micronutrients Iron (Fe) and Zinc (Zn), the gluten storage proteins α-gliadins and high molecular weight glutenin subunits (HMW-GS), and the secondary metabolite phenolics. Generally, the timopheevii lineage has been neglected to date regarding grain quality studies. Thus, the timopheevii lineage should be subject to grain quality research to explore the full diversity of the wheat gene pool. # Introduction Wheat is one of the world's most important crops. It provides 19% of human calorie intake and 21% of protein intake [bib_ref] Crops that feed the world 10 past successes and future challenges to..., Shiferaw [/bib_ref]. Hexaploid bread wheat (Triticum aestivum L., 2n = 6x = 42, BBA u A u DD) accounts for about 90% of wheat production, and tetraploid pasta wheat (Triticum durum Desf., 2n = 4x = 28, BBA u A u ) is the 10th most important staple crop (International Grains Council (2020). World Grain Statistics 2016). The two economically most important wheat species have different levels of ploidy. This is due to the evolutionary history, in which the process of domestication plays a predominant role [bib_ref] Genome plasticity a key factor in the success of polyploid wheat under..., Dubcovsky [/bib_ref]. Domesticated plants fulfilled the requirements of human cultivation and dietary preference. The main characteristic of domesticated cereals is the loss of the ability to survive independently in the wild [bib_ref] Evolution of crop species: genetics of domestication and diversification, Meyer [/bib_ref] [bib_ref] The nature of selection during plant domestication, Purugganan [/bib_ref]. The center of wheat diversity and initial domestication lies in the Fertile Crescent. The evolutionary history of wheat is characterized by the allo-polyploidization of the originally diploid Triticum and Aegilops species, which led to the emergence of tetra-and hexaploid wheat species. Human cultivation of the tetraploid wild emmer Triticum dicoccoides (Körn. ex. Aschers. & Graebner) Schweinf. (2n = 4x = 28, BBA u A u ) resulted in the emergence of the domesticated Triticum dicoccon Schübl. (2n = 4x = 28, BBA u A u ) and later T. durum. The domesticated tetraploid species as well as T. aestivum, of which no direct hexaploid wild relative has yet been identified, expanded their cultivation area by human migration and trade [bib_ref] Genetic diversity, evolution and domestication of wheat and barley in the fertile..., Kilian [/bib_ref]. Migration from their center of diversity and continuous breeding efforts reduced the genetic diversity of T. aestivum and T. durum [bib_ref] Seed banks and molecular maps: unlocking genetic potential from the wild, Tanksley [/bib_ref] [bib_ref] Plant domestication, a unique opportunity to identify the genetic basis of adaptation, Ross-Ibarra [/bib_ref]. Currently, climatic conditions are changing, posing a threat to the productivity of the cultivated wheats [bib_ref] Temperature increase reduces global yields of major crops in four independent estimates, Zhao [/bib_ref] [bib_ref] Rising temperatures and increasing demand challenge wheat supply in Sudan, Iizumi [/bib_ref] [bib_ref] Adverse weather conditions for European wheat production will become more frequent with..., Trnka [/bib_ref]. The wild and domesticated relatives of wheat are a valuable source of beneficial genetic diversity 1 3 that can be used for crop improvement [bib_ref] Adapting agriculture to climate change: a global initiative to collect, conserve, and..., Dempewolf [/bib_ref] [bib_ref] Exploring natural selection to guide breeding for agriculture, Henry [/bib_ref] [bib_ref] Genomics of crop wild relatives: expanding the gene pool for crop improvement, Brozynska [/bib_ref] [bib_ref] Crop science special issue: adapting agriculture to climate change: a walk on..., Kilian [/bib_ref] [bib_ref] Introducing beneficial alleles from plant genetic resources into the wheat germplasm, Sharma [/bib_ref]. Wild relatives of wheat are well adapted to their natural habitats, which can be harsh, so they carry useful traits for abiotic stress tolerance [bib_ref] Exploring natural selection to guide breeding for agriculture, Henry [/bib_ref] [bib_ref] Wheat: mechanisms and genetic means for improving heat tolerance, Singh [/bib_ref]. Their genetic diversity has also been exploited for their resistance to various pests, e.g., hessian fly [bib_ref] New durum wheat with Hessian fly resistance from Triticum araraticum and T..., Nsarellah [/bib_ref] and diseases, such as powdery mildew [bib_ref] A CNL protein in wild emmer wheat confers powdery mildew resistance, Li [/bib_ref] and leaf rust [bib_ref] Identification of new leaf rust resistance loci in wheat and wild relatives..., Fatima [/bib_ref] [bib_ref] Discovery and characterisation of a new leaf rust resistance gene introgressed in..., Narang [/bib_ref]. These beneficial traits can be incorporated into modern wheat cultivars via (pre-)breeding programs [bib_ref] Introducing beneficial alleles from plant genetic resources into the wheat germplasm, Sharma [/bib_ref] and thus illustrate the value of this genepool for securing the future cultivation of wheat. In order to harness and conserve the beneficial diversity of wild relatives, genebanks play an important role as ex situ conversation sites [bib_ref] Adapting agriculture to climate change: a global initiative to collect, conserve, and..., Dempewolf [/bib_ref] [bib_ref] Crop science special issue: adapting agriculture to climate change: a walk on..., Kilian [/bib_ref]. An overview of ex situ Triticum collections can be found in [bib_ref] Introducing beneficial alleles from plant genetic resources into the wheat germplasm, Sharma [/bib_ref] Due to wheat's role as a staple, the quality of the grain is of great importance. Its daily consumption in many parts of the world makes it a major source for calorie intake, but it also provides micronutrients and vitamins. Breeding for energy yield and grain quality for purposes such as baking has long been a major goal in wheat breeding and enabled food security for a growing population. However, the focus on micronutrients and allergenicity has only been a recent trend in wheat breeding. Especially in less diversified diets, a low concentration of micronutrients in wheat appears to be a major problem. This leads to micronutrient deficiencies ("hidden hunger"), especially for the crucial micronutrients iron (Fe) and zinc (Zn) [bib_ref] Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: Principles,..., Graham [/bib_ref] [bib_ref] Genetic perspectives on crop domestication, Graham [/bib_ref] [bib_ref] Biofortification, biodiversity and diet: a search for complementary applications against poverty and..., Johns [/bib_ref]. The main symptom of Fe deficiency is anemia, and Zn deficiency can affect growth and development [bib_ref] Global impacts of human mineral malnutrition, Stein [/bib_ref] [bib_ref] A review of phytate, iron, zinc, and calcium concentrations in plant-based complementary..., Gibson [/bib_ref]. To combat hidden hunger, one commonly used approach is biofortification (= enrichment of micronutrients) of crops [bib_ref] Biofortification, biodiversity and diet: a search for complementary applications against poverty and..., Johns [/bib_ref]. Crop biofortification can be achieved through agronomic strategies such as foliar fertilization or via various breeding methods such as introgression. Some wild relatives of wheat accumulate micronutrients very efficiently in the grain and offer high genetic diversity for this trait [bib_ref] Genomewide association mapping of grain micronutrients concentration in aegilops tauschii, Arora [/bib_ref] [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref] [bib_ref] Aegilops kotschyi and aegilops tauschii as sources for higher levels of grain..., Chhuneja [/bib_ref] [bib_ref] Genetic diversity for grain nutrients in wild emmer wheat: potential for wheat..., Chatzav [/bib_ref]. Therefore, they can be used as a breeding resource for biofortified crops [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref] [bib_ref] Evaluation and utilization of Aegilops and wild Triticum species for enhancing iron..., Rawat [/bib_ref]. Wheat is consumed in various forms, for example as bread, pasta, bulgur and couscous. An important qualitydetermining characteristic is gluten, which comprises a group of seed storage proteins composed of glutenins and gliadins. While the former is very important for dough quality and elasticity, the latter is responsible for viscosity [bib_ref] Chemistry of gluten proteins, Wieser [/bib_ref]. However, gliadins can trigger an autoimmune disease called celiac disease (CD) (Biesiekierski 2017). Currently, the only strategy for CD patients is to avoid wheat products. The wheat genepool may harbor diversity that can help to combine improved dough quality properties of glutenin with less toxic and non-immunogenic gliadin variants for CD-safe wheat. Phenolics are bioactive components in the grain with promising health benefits for the consumer. Phenolic compounds can be grouped into phenolic acids, flavonoids, stilbenes, coumarines, lignans and tannins. Phenolic acids are the most abundant component in wheat and are predominantly stored in the aleurone layer and the bran [bib_ref] Importance of insoluble-bound phenolics to antioxidant properties of wheat, Liyana-Pathirana [/bib_ref]. The domesticated einkorn and emmer were assumed to be enriched in phenolic acid content [bib_ref] Phytochemical quantification and total antioxidant capacities of emmer (Triticum dicoccon Schrank) and..., Serpen [/bib_ref] [bib_ref] The effect of species and cultivation year on phenolic acids content in..., Barański [/bib_ref] , and thus, wild wheats could be a valuable source for the enrichment of phenolic acids in the whole wheat grain. This review provides an overview of the state of research on grain quality traits in the wheat genepool, including micronutrients, gluten and phenolics. Research on grain quality parameters in wheat relatives is summarized and assessed in the context of evolutionary history. ## The process of domestication Humans selected wild plants with favorable characteristics for cultivation and preparation to ensure food security. This process genetically transformed the plants into a domesticated plant that is dependent on human care and thus loses its ability to survive in the wild [bib_ref] The molecular genetics of crop domestication, Doebley [/bib_ref]. The modified plants showed a distinctive morphology that is referred to as the domestication syndrome . The domestication syndrome evolved from the selection of wild plants for higher germination rates and easier harvesting [bib_ref] The nature of selection during plant domestication, Purugganan [/bib_ref]. The most common traits are the loss of seed shattering, increased seed size and alteration of the reproductive organs [bib_ref] The molecular genetics of crop domestication, Doebley [/bib_ref] [bib_ref] Plant domestication, a unique opportunity to identify the genetic basis of adaptation, Ross-Ibarra [/bib_ref] [bib_ref] Evolution of crop species: genetics of domestication and diversification, Meyer [/bib_ref]. These domestication traits evolved through a combination of conscious and unconscious selection. Humans actively carried out conscious selection by selecting a favored trait and thus forwarded this trait into the next generation. Unconscious selection, on the other hand, favored the adaptability to the new agro-ecological environment shaped by human farming systems. As a result, the plant improved its performance in the field and was chosen for further breeding [bib_ref] Unconscious selection and the evolution of domesticated plants, Zohary [/bib_ref]. In the beginning, wild and cultivated plants grew close to each other, which led to the exchange of genes via cross-pollination and gene flow [bib_ref] The role of gene flow and chromosomal instability in shaping the bread..., Przewieslik-Allen [/bib_ref] [bib_ref] Exome sequencing highlights the role of wild-relative introgression in shaping the adaptive..., He [/bib_ref]. This scenario still occurs today in the centers of origin of crops. However, human intervention has morphologically and genetically modified 1 3 the wild plants, resulting in a newly domesticated species [bib_ref] The genetics and genomics of plant domestication, Kantar [/bib_ref]. In addition to selection for domestication traits, selection for plant improvement further differentiated the domesticated species. This process is called diversification phase and involves the adaption of plants to new environments and selection for quality traits that differed individually for each crop [bib_ref] Evolution of crop species: genetics of domestication and diversification, Meyer [/bib_ref]. As a result, the differently pronounced selection pressure led to further divergence within the species. Genetic alteration and introduction into new habitats led to adaptation of a domesticated species. In summary, the domestication and improvement phase increased diversity in terms of the number of newly emerged domesticated taxa. However, it is important to focus on the initial situation of domestication. The onset of domestication occurred independently in different areas around the world at different times, but the traits of interest were similar [bib_ref] The nature of selection during plant domestication, Purugganan [/bib_ref] [bib_ref] Evolution of crop species: genetics of domestication and diversification, Meyer [/bib_ref]. From the broad wild diversity that was available to humans, only a few plants were selected and used as founder crops [bib_ref] Founder effect in crop-plant evolution, Ladizinsky [/bib_ref]. Consistent cultivation and selection within these founder crops led to a genetic bottleneck that excluded many wild alleles from the domestication process [bib_ref] The molecular genetics of crop domestication, Doebley [/bib_ref] [bib_ref] How to make a domesticate, Stetter [/bib_ref]. The diversity retrieved during the domestication and improvement phase cannot compensate for the reduced diversity at the beginning [bib_ref] The impact of genetic changes during crop domestication, Smýkal [/bib_ref]. The founder crop (population) experienced high selection pressure on domestication traits. Increased selection led to selective sweeps that are reflected in a reduced diversity of domestication loci compared to other loci [bib_ref] The impact of genetic changes during crop domestication, Smýkal [/bib_ref] [bib_ref] Durum wheat genome highlights past domestication signatures and future improvement targets, Maccaferri [/bib_ref] [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. Most early domesticates were annuals because the fixation of traits through an annual selection cycle was more successful and faster compared to perennials. Thus, selection pressure on desired traits accelerated the loss of diversity in the corresponding loci and reduced overall diversity [bib_ref] Durum wheat genome highlights past domestication signatures and future improvement targets, Maccaferri [/bib_ref]. ## Domestication history of wheat and its resulting diversity Wheat's allo-polyploidy accelerated its wider distribution, as polyploid species show a better adaption rate to new environments and outperform their ancestors with a lower degree of ploidy in agronomic traits [bib_ref] Genome plasticity a key factor in the success of polyploid wheat under..., Dubcovsky [/bib_ref]. Therefore, farmers favored them, which led to a larger cultivation area and facilitated their spread from the Fertile Crescent across the world. ## Diploid wheats Diploid wheats (AA) belong to the oldest cultivated plant species. Wild representatives of the diploid wheat group are Triticum boeoticum Boiss (2n = 2x = 14, AA) and Triticum urartu Thumanjan ex Gandilyan (2n = 2x = 14, A u A u ). Triticum monococcum L. (2n = 2x = 14, AA) is the domesticate of T. boeoticum. Together, they form the einkorn group of wheat. The Karacadağ and Kartal-Karadağ mountains in Turkey have been identified as their center of domestication [bib_ref] Site of einkorn wheat domestication identified by DNA fingerprinting, Heun [/bib_ref] [bib_ref] Molecular diversity at 18 loci in 321 wild and 92 domesticate lines..., Kilian [/bib_ref]. From here, einkorn migrated east (Armenia, Georgia, Iran) and west via the Bosporus (Greece and toward Central Europe). Another route involved maritime transport to the Maghreb and the Iberian Peninsula. These routes are supported by archaeological evidence, but also the population structure of the einkorn genepool that was shaped by the migration routes (reviewed in: Zaharieva and Monneveux 2014; [bib_ref] Geographic differentiation of domesticated einkorn wheat and possible Neolithic migration routes, Brandolini [/bib_ref] [fig_ref] Figure 1: Origin and spread of domesticated einkorn wheat T [/fig_ref]. T. urartu is phenotypically similar to T. boeoticumbut can be distinguished genetically [bib_ref] Molecular markers based on LTR retrotransposons BARE-1 and Jeli uncover different strata..., Konovalov [/bib_ref]. T. urartu represents the A genome donor of tetra-and hexaploid wheats [bib_ref] Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex..., Huang [/bib_ref]. T. urartu populations occur in the Fertile Crescent and are found in Lebanon, Turkey, Iraq, Iran and Transcaucasia. This geographic distribution also shaped the genetic diversity of T. urartu by forming groups according to bioclimatic and topographic criteria [bib_ref] Molecular diversity and landscape genomics of the crop wild relative Triticum urartu..., Brunazzi [/bib_ref]. ## Tetraploid wheats Hybridization between T. urartu and a close relative of Aegilops speltoides Tausch (2n = 2x = 14, BB/GG), a member of the Sitopsis section in Aegilops, initiated the formation of the tetraploid wild wheat, T. dicoccoides. This wild emmer wheat further evolved into the domesticated T. dicoccon. However, the emmer lineage is not the only tetraploid wild wheat. Triticum araraticum Jakubz. (2n = 4x = 28, GGA u A u ) is also a descendant of an independent hybridization between T. urartu and Ae. speltoides. Although both tetraploid wheat lineages share potentially the same parental species, they differ in the structure of the genome and time of origin [bib_ref] GAA)n microsatellite as an indicator of the A genome reorganization during wheat..., Adonina [/bib_ref]. Diversity analysis clearly showed a shared ancestry of Ae. speltoides and tetra-and hexaploid wheats. Ae. speltoides shared nuclear and cytoplasmic loci with T. dicoccoides and T. araraticum, which differed between the two wild tetraploid wheat species. Therefore, it was proposed that both genomes were derived (at least in parts) from Ae. speltoides as a maternal donor [bib_ref] Independent wheat B and G genome origins in outcrossing Aegilops progenitor haplotypes, Kilian [/bib_ref] [bib_ref] Molecular phylogeny of the genus Triticum L, Golovnina [/bib_ref]. However, the G genome of T. araraticum shows a closer relationship with Ae. speltoides, while the B genome donor is more distant to Ae. speltoides [bib_ref] Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex..., Huang [/bib_ref] [bib_ref] Reconciling the evolutionary origin of bread wheat (Triticum aestivum), El Baidouri [/bib_ref] [bib_ref] Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae, Bernhardt [/bib_ref]. ## 3 ## Domestication and cultivation of the bbaa lineage and the hexaploid bbaadd Molecular analysis of T. dicoccoides revealed two subgroups with distinct geographic origin. The southern Levant subgroup included genotypes from Israel, Palestine, Jordan, Syria and Lebanon. Accessions belonging to the other group were associated with the Central Eastern Fertile Crescent extending from Turkey to Iraq and Iran [bib_ref] The structure of wild and domesticated emmer wheat populations, gene flow between..., Luo [/bib_ref] [bib_ref] A reconsideration of the domestication geography of tetraploid wheats, Ozkan [/bib_ref] [bib_ref] Durum wheat genome highlights past domestication signatures and future improvement targets, Maccaferri [/bib_ref]. Some studies suggested a monophyletic origin of domesticated emmer from within the Central Eastern group. However, archaeological data indicate early cultivation of emmer in the Southern Levant (reviewed in: . Recent analysis suggested a multi-regional origin by showing that domesticated emmer contains alleles from both T. dicoccoides populations. It was concluded that wild emmer was pre-cultivated in the southern Levant and met wild emmer from the Central Eastern group through migration northwards, where it was eventually domesticated [bib_ref] Multiregional origins of the domesticated tetraploid wheats, Oliveira [/bib_ref]. T. dicoccon spread in all directions via various routes. Europe was reached via the Bosporus, the Balkans and the Iberian Peninsula. Africa was reached via the southern Levant and the Arabian Peninsula, and the spread to Asia was via Iran (reviewed in: Martínez-Moreno et al. 2020; [bib_ref] Chromosomal passports provide new insights into diffusion of emmer wheat, Badaeva [/bib_ref] [fig_ref] Figure 2: Origin and spread of domesticated emmer and durum wheat [/fig_ref]. T. durum is currently the economically most important cultivated species from the tetraploid BBAA genepool. Durum wheat most likely originated in the eastern Mediterranean region [bib_ref] The structure of wild and domesticated emmer wheat populations, gene flow between..., Luo [/bib_ref] [bib_ref] Genetic diversity within a global panel of durum wheat (triticum durum) landraces..., Kabbaj [/bib_ref] , which is supported by a high genetic diversity in durum wheat accessions from the Central Fertile Crescent . However, its evolution has not yet been fully elucidated. Phylogenetic analysis of wild and domesticated emmer, durum wheat landraces and durum cultivars revealed a close relationship between durum wheat landraces and domesticated emmer from the southern Levant. This suggests a rather linear evolution, in which increased selection and improvement on T. dicoccon facilitated the emergence of T. durum landraces. Moreover, modern durum wheat cultivars showed the closest relationship to durum wheat landraces from North Africa and Transcaucasia (Maccaferri et al. 2019). In contrast, exome sequencing data of the wheat genepool identified T. dicoccoides as a direct donor to T. dicoccon and T. durum, suggesting an entangled evolution [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. From its potential center of origin, durum wheat spread widely following similar expansion routes as T. dicoccon [bib_ref] Dispersal of durum wheat [Triticum turgidum L. ssp. turgidum convar. durum (Desf.)..., Moragues [/bib_ref] [bib_ref] Genetic diversity within a global panel of durum wheat (triticum durum) landraces..., Kabbaj [/bib_ref] ; Martínez-Moreno et al. 2020) [fig_ref] Figure 2: Origin and spread of domesticated emmer and durum wheat [/fig_ref]. The emmer lineage evolved further via another hybridization event, which gave rise to the hexaploid wheats. Most likely, domesticated tetraploid wheat and Aegilops tauschii Cosson (2n = 2x = 14, DD) hybridized in the area of northeastern Iran and the southwestern Caspian Sea, giving rise to the hexaploid bread wheat, T. aestivum [bib_ref] Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of..., Petersen [/bib_ref] [bib_ref] Genetics and geography of wild cereal domestication in the near east, Salamini [/bib_ref] [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. The highest genetic diversity for hexaploid bread wheat was found in the Near and Middle East, which is probably the center of diversity . Hexaploid bread wheat is based on a founder race that further evolved into [bib_ref] Site of einkorn wheat domestication identified by DNA fingerprinting, Heun [/bib_ref] [bib_ref] Molecular diversity at 18 loci in 321 wild and 92 domesticate lines..., Kilian [/bib_ref] ; arrows indicate T. monococcum's dispersal pathways (Zaharieva and Monneveux 2014; [bib_ref] Geographic differentiation of domesticated einkorn wheat and possible Neolithic migration routes, Brandolini [/bib_ref] 1 3 two hexaploid wheat genepools [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. Moreover, the wheat population can be divided into a western and far eastern subpopulation, the latter resembling the ancient wheat due to less introgression from Ae. tauschii . Polyploidization limited the initial diversity of the new species, as only a small number of genotypes were potentially involved in its emergence [bib_ref] Genome plasticity a key factor in the success of polyploid wheat under..., Dubcovsky [/bib_ref] [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. However, due to introgression from T. dicoccoides into the hexaploid wheat genome, some diversity was retained [bib_ref] Exome sequencing highlights the role of wild-relative introgression in shaping the adaptive..., He [/bib_ref]. ## Domestication and cultivation of the wheat g genome containing lineage In contrast to the emmer lineage, the timopheevii lineage with the genomic constitution GGAA received less attention. The evolutionary history of the GGAA wheat genepool is complex, as several chromosomal rearrangements were involved . The wild ancestral form of the timopheevii lineage is T. araraticum. Northern Iraq is the center of diversity and origin of T. araraticum [bib_ref] The chloroplast view of the evolution of polyploid wheat, Gornicki [/bib_ref] [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] [fig_ref] Figure 3: Origin and spread of GGAA wheat [/fig_ref]. T. araraticum comprises two subgroups. One subgroup (ARA-0) is widespread, while the other (ARA-1) was only found in south-eastern Turkey and north-western Syria . Triticum timopheevii (Zhuk.) Zhuk (2x = 4n = 28, GGA u A u ) is the domesticate of T. araraticum. The origin of T. timopheevii s.str. (found in Georgia) remains unclear, but was probably introduced from Turkey [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref]. [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] also discuss the potential sister-group relationship between the Georgian T. timopheevii s.str. and the prehistoric and widespread T. timopheevii s.l. ('New Glume Wheat'), with the oldest known records being of Turkish origin. The mixed cultivation of T. monococcum and T. timopheevii in western Georgia facilitated hybridization between the two. This event resulted in the hexaploid Triticum zhukovskyi Men. & Er. (2n = 6x = 42, GGA u A u A m A m ) [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] [fig_ref] Figure 3: Origin and spread of GGAA wheat [/fig_ref]. However, [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref] suggested a hybridization between T. araraticum and T. boeoticum for the formation of T. zhukovskyi. [bib_ref] Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of..., Petersen [/bib_ref] [bib_ref] Genetics and geography of wild cereal domestication in the near east, Salamini [/bib_ref] [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref] 1 3 ## Micronutrients in wheat wild relatives Iron (Fe) and Zinc (Zn) deficiency are major forms of micronutrient disorders in human diets relying on one major crop. Fe deficiency leads to anemia and insufficient Zn uptake impairs various essential functions of the body including growth, development and the immune system [bib_ref] Global impacts of human mineral malnutrition, Stein [/bib_ref]. The large wheat genepool described earlier may harbor beneficial genetic variation for an increased accumulation of micronutrients in the edible parts of the wheat crop. Furthermore, the evolutionary pattern can be used to identify trends and underutilized species for biofortification. ## Wheat wild relatives show high enrichment and variation for fe and zn in the grain Aegilops species and the einkorn and emmer lineage were of interest for the grain micronutrient concentration and the variation. The wild species harbored a high variation for Zn grain concentration, whereas Aegilops species showed elevated Fe grain concentration. Domesticated einkorn wheat forms a valuable source for biofortification, especially for Zn [bib_ref] Environmental and genotypic influences on trace element and mineral concentrations in whole..., Erba [/bib_ref]. In addition, high values of Fe and Zn were consistently found in T. monococcum accessions across different locations [bib_ref] Environmental and genotypic influences on trace element and mineral concentrations in whole..., Erba [/bib_ref]. Therefore, an underlying genetic component is likely. Compared to T. monococcum, the variation in Zn and Fe concentrations was higher in T. boeoticum in the studies comparing the wild and domesticated forms [fig_ref] Figure 1: Origin and spread of domesticated einkorn wheat T [/fig_ref]. This underlines the greater potential for genetic variation in crop wild relatives. Furthermore, grains of T. zhukovskyi could be potentially enriched in Zn, due to the fact that it contains the A genome of T. monococcum. Among the few studies available, most focused on T. monococcum and T. boeoticum and excluded T. urartu, the actual A genome donor of emmer, durum, bread wheat and timopheevii wheat. Because of this ancestry, exploring the micronutrient content and accumulation of T. urartu might be valuable for the biofortification of its descendants. Diploid wheats have been less extensively investigated than their tetraploid relatives from the emmer lineage. One reason for this lack of interest could be their inferior agricultural performance or the challenges in introgressing beneficial alleles into bread wheat. Thus, there is still a research gap that needs to be explored. The same is true for Aegilops accessions, which have been investigated only sporadically, although some accessions could be a promising resource for biofortification. Compared to cultivated bread wheat, Ae. tauschii and Ae. kotschyi showed higher grain Fe and Zn concentrations [bib_ref] Aegilops kotschyi and aegilops tauschii as sources for higher levels of grain..., Chhuneja [/bib_ref] [bib_ref] Evaluation and utilization of Aegilops and wild Triticum species for enhancing iron..., Rawat [/bib_ref]. Tetraploid wild emmer wheat (T. dicoccoides) has always received attention as this species provides a valuable source of genetic variation in terms of accumulation of micronutrients [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref] [bib_ref] Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat..., Peleg [/bib_ref]. Higher accumulation of Fe and Zn in T. dicoccoides compared to T. durum was also consistent under water deficient conditions [bib_ref] Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat..., Peleg [/bib_ref]. Compared to modern wheat cultivars, wild emmer wheat showed a higher variation in Zn and Fe concentration [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref]. Comparing the variation between Fe and Zn in wild diploid T. boeoticum and wild tetraploid emmer wheat, the amplitude for Zn was higher than for Fe [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref]. Studies that analyzed iron and/or zinc in wild wheat accessions were summarized to calculate an overall mean (mg/kg) . Further, the mean value of species analyzed in at least two independent studies was used for comparison between species. Due to the small number of studies, the median and the individual values were visualized [fig_ref] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies... [/fig_ref]. Aegilops accessions showed higher iron accumulation compared to Triticum taxa across different studies [fig_ref] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies... [/fig_ref]. However, the highest value [bib_ref] The chloroplast view of the evolution of polyploid wheat, Gornicki [/bib_ref] [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] ; red circle is the domestication region of T. timopheevii [bib_ref] Genetic diversity and origin of timopheevi wheat inferred by chloroplast DNA fingerprinting, Mori [/bib_ref] [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] ; orange circle indicates the hybridization site of T. zhukovskyi [bib_ref] A Whole genome DArTseq and SNP analysis for genetic diversity assessment in..., Badaeva [/bib_ref] ## Table 1 Summary of studies in which more than one genotype of the respective species was examined for Fe and Zn concentration in the grain for grain Fe concentration in T. boeoticum was in the same range as the median Fe concentration in Ae. kotschyi and Ae. tauschii. Such clear differentiation was not present for grain Zn concentration across species. The variation in grain Zn concentration was higher, as shown by the wider distribution of the mean values. This variation was pronounced in T. dicoccoides, which showed the highest median for grain Zn concentration [fig_ref] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies... [/fig_ref]. This confirms the observations that the variation is higher for Zn accumulation than for Fe. It is noteworthy that this enrichment and variation found in wild emmer has not been passed on and confirmed in its economically important progeny T. durum [fig_ref] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies... [/fig_ref]. ## Factors influencing the fe and zn concentration in the grain How, if and to what extend the micronutrient concentration in the grain is influenced by external factors and the phenotype, e.g., grain size, is contradictory to date. Some authors argue that enhanced micronutrient density may come at the expense of agronomic performance. Progress in breeding for higher yields may have diluted the concentration of micronutrients in the grain, which is referred to as the dilution effect or reversible concentration effect in small grains [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref]. This hypothesis is supported by observations of negative correlations between yield and grain Zn concentrations [bib_ref] Genetic variability and stability of grain magnesium, zinc and iron concentrations in..., Oury [/bib_ref]. Negative correlation also occurred between the year of release of the respective variety and its micronutrient density [bib_ref] Variation in mineral micronutrient concentrations in grain of wheat lines of diverse..., Zhao [/bib_ref] , suggesting that the breeding for high yield negatively affects micronutrient concentrations. This observation was more pronounced for Zn compared to Fe [bib_ref] Genetic variability and stability of grain magnesium, zinc and iron concentrations in..., Oury [/bib_ref]. On the other hand, some studies found no correlation between seed size and micronutrient concentration, calling into question the hypothesis of the dilution effect [bib_ref] Genomewide association mapping of grain micronutrients concentration in aegilops tauschii, Arora [/bib_ref] [bib_ref] Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations, Distelfeld [/bib_ref]. The micronutrient content per seed was analyzed in addition to the concentration. Seeds with the highest concentration also had the highest content per seed due to high seed weight and size [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref] [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref]. This clearly refutes the dilution hypothesis. In conclusion, the results concerning the relationship between agronomic performance and micronutrient density are controversial, suggesting that both components contribute to micronutrient density. To identify the traits associated with grain micronutrient concentrations, correlations with other grain characteristics were analyzed. Agronomic management practices can help to increase micronutrient concentrations in the grain. Plants grown under nitrogen-sufficient conditions showed enriched micronutrients levels compared to nitrogen-deficient conditions [bib_ref] Breeding for trace minerals in Wheat, Monasterio [/bib_ref]. Furthermore, higher grain protein content was also associated with higher micronutrients [bib_ref] Genetic diversity for grain nutrients in wild emmer wheat: potential for wheat..., Chatzav [/bib_ref] [bib_ref] Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat..., Peleg [/bib_ref]. Fe and Zn are preferentially stored in the aleurone layer and the embryo. At the same time, the embryo contains high protein contents, which can explain the positive associations . As nutrients are translocated from the leaves to the grain during grain filling, nutrient concentrations (N, Fe, Zn) in the flag leaf were correlated with grain concentrations [bib_ref] Evaluation and utilization of Aegilops and wild Triticum species for enhancing iron..., Rawat [/bib_ref]. These findings suggest that grain protein is a sink for micronutrients and explain why bread wheat shows lower micronutrient concentration than durum wheat . In conclusion, an adequate supply of nitrogen can help to exploit the full potential of micronutrient accumulation in the grain. However, there is also another genetic component that supports wild wheats in the enrichment of micronutrients in the grain. Genetic properties of micronutrient accumulation in wheat wild relatives Micronutrient uptake and translocation are quantitatively controlled and inherited traits [bib_ref] Genomewide association mapping of grain micronutrients concentration in aegilops tauschii, Arora [/bib_ref] that can be genetically dissected by genome mapping. Therefore, Recombinant Inbred Lines (RIL) from two domesticated einkorn lines were developed byin order to gain insights into the genetics of micronutrient uptake. They found a promising Quantitative Trait Loci (QTL) on the short arm of chromosome 5 that contributes micronutrient accumulation. Chromosome 5A also harbored favorable QTLs for Fe and Zn micronutrient concentrations in a tetraploid RIL population derived from durum wheat and wild emmer. The authors stated that the wild alleles increased micronutrient accumulation [bib_ref] Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat x..., Peleg [/bib_ref]. Furthermore, chromosome 6B was detected as a promising gene source conferring high micronutrient concentrations in grains [bib_ref] Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat x..., Peleg [/bib_ref] [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref] [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref]. RIL lines containing chromosome 6B of T. dicoccoides had an enriched Zn concentration in the grain [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref] [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref]. Independent of this finding, the QTL GPC-B1 on the short arm of chromosome 6B of T. dicoccoides was associated with elevated grain protein concentration [bib_ref] Mapping gene(s) for grain protein in tetraploid wheat (Triticum turgidum L) using..., Joppa [/bib_ref]. GPC-B1 showed a direct influence on chlorophyll degradation of flag leaves, thus accelerating senescence, and had pleiotropic effects on grain protein [bib_ref] The high grain protein content gene Gpc-B1 accelerates senescence and has pleiotropic..., Uauy [/bib_ref]. Consequently, the transportation mechanism of nitrogen from leaves to grains was investigated for its role in grain micronutrient content. Recombinant chromosome substitution lines carrying the GPC-B1 allele from wild emmer wheat showed increased Zn and Fe concentration in the grain compared to the GPC-B1 allele from durum wheat [bib_ref] Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations, Distelfeld [/bib_ref]. The QTL was narrowed down to the allele NAM-B1, which encodes a NAC transcription factor . Reduced expression of NAM-B1 resulted in plants with lower levels of protein, Zn and Fe in the grain, but higher levels in the flag leaf. This proved the role of NAM-B1 in remobilization of micronutrients and nitrogen and further explained the observed positive correlation between these components described above. Only wild wheat species possessed the functional NAM-B1 genes (GPC-B1 gene, used synonymously), leading to higher accumulation of micronutrients compared to T. durum and T. aestivum, where the allele is non-functional. Hexaploid wheat contains two functional homologous genes on chromosomes 6A and 6D . This genetic alteration occurred during wheat evolution, emphasizing the benefit of wild relatives. Therefore, the genomes of wheat relatives were explored for similar loci. Orthologues of NAM-B1 were identified in T. timopheevii, Ae. speltoides, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. sharonensis [bib_ref] Characteristics and polymorphism of NAM gene from Aegilops section sitopsis species, Hu [/bib_ref] [bib_ref] Variation and their relationship of NAM-G1 gene and grain protein content in..., Hu [/bib_ref]. Apart from some Single Nucleotide Polymorphisms (SNP), the T. timopheevii NAM-G1 orthologue resembled the T. dicoccoides NAM-B1 gene in promoting grain protein content and was therefore considered as a putative target for micronutrient enrichment. However, the follow-up study by [bib_ref] Zn and Fe concentration variations of grain and flag leaf and the..., Hu [/bib_ref] showed no association between NAM-G1 expression levels and grain Fe and Zn concentration. Consequently, they proposed a quantitatively controlled trait for micronutrient concentrations in grain rather than just NAM-G1 as a single gene causing the higher micronutrient content . Another explanation for this finding could be that the allele does not function properly due to T. timopheevii's domestication status. To confirm this, a study with its wild ancestor T. araraticum would be required. Additionally, Ae. speltoides or an extinct close relative of it participated as donors for the B and G genome. However, different genoytpes were involved in the independent polyploidization events of the emmer and timopheevii lineage. Since the functional NAM gene is found on the B1 genome, it might be non-functional in G1, thus explaining the lower micronutrient concentration in T. timopheevii. ## Regulatory role of the gpc gene The physiological implications of this wild gene in hexaploid wheats were studied to elucidate its role in metal homeostasis. The pre-and post-anthesis phases are the most critical for micronutrient accumulation. Fe and Zn are not directly transferred into the grain. Initially, they are stored in the leaves, where they play an important role in the function of essential enzymes in mitochondria and plastids [bib_ref] Biofortification and bioavailability of Zn, Fe and Se in wheat: present status..., Gupta [/bib_ref]. GPC is expressed during early senescence to prepare the plant for subsequent physiological processes [bib_ref] Effect of the down-regulation of the high Grain Protein Content (GPC) genes..., Cantu [/bib_ref]. Transcriptome analysis showed that under a high GPC expression levels, genes involved in transport, protein metabolism and catabolic/catalytic processes are also induced [bib_ref] Effect of the down-regulation of the high Grain Protein Content (GPC) genes..., Cantu [/bib_ref]. To explain the highly efficient translocation of micronutrients from flag leaf to grain enabled by GPC, Fe and Zn transporters were analyzed under putative GPC expression control. A Heavy Metal ATPase-like (HMA-like) was induced in the presence of GPC but was downregulated in gpc knock out mutants. HMAs and HMA-like transporters enable the transfer of micronutrients from the chloroplast to the cytoplasm. From here, the released nutrients must reach the phloem. Genes from the ZIP family, which include transporters for Fe (IRT = iron regulated transporter) and Zn (ZRT = zinc regulated transporter), formed the majority of upregulated genes under GPC influence. For phloem transport, Fe and Zn must be chelated. In this context, a gene involved in the biosynthesis of the phytosiderophores (PS) nicotianamine (NA), nicotianamine aminotransferase (NAAT), was found at a higher level [fig_ref] Figure 5: Regulatory role of the GPC-encoded NAC transcription factor for micronutrient translocation [/fig_ref]. In line with this finding, effective uptake and transportation via PS have also been suggested to be responsible for the higher micronutrient concentration in Aegilops species compared to Triticum species [bib_ref] Identification of Aegilops species with higher production of phytosiderophore and iron and..., Neelam [/bib_ref]. A genome-wide association study with the D genome donor Ae. tauschii confirmed the role of PSs by identifying marker-trait associations for iron concentrations. A significant locus was found near a gene involved in PS secretion [bib_ref] Genomewide association mapping of grain micronutrients concentration in aegilops tauschii, Arora [/bib_ref]. This shows that a subset of genes is controlled by the transcription factor encoding GPC gene, which facilitates the translocation of micronutrients. It is a unique example on how valuable wild alleles can be used for biofortification. However, since the GPC was not solely responsible for the accumulation of micronutrients in the grain, other genes need to be explored. The identification of the functional GPC gene helps to understand the observed correlations between grain protein, yield and micronutrients explained above. First, it provides a plausible explanation for T. dicoccoides being such a promising source for higher grain micronutrient concentrations due to the functional GPC-B1 allele. However, it remains unclear why there is such a high variation for Zn and Fe between T. dicoccoides accessions and how values as low as 15 mg/kg for Fe and 14 mg/kg for Zn can occur despite the presence of these functional alleles. One explanation could be a pronounced environmental influence, e.g., low micronutrient concentration in the soil. However, the wild GPC-B1 alleles showed a stable contribution across different environments and no interaction between genotype and environment [bib_ref] Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations, Distelfeld [/bib_ref]. Either, these accessions lack the gene, perhaps due to adaption effects to different areas, or other genes associated with micronutrient accumulation may be equally important, which remain to be identified. Second, the GPC gene might be involved in mechanisms leading to the dilution effect suggested earlier. Domesticated varieties contain functional orthologues of the GPC allele only on the A and D genomes, which are less efficient in micronutrient translocation from the flag leaf to the grain than the copy on the B genome. Since the GPC-mediated translocation is more efficient for Fe than for Zn [bib_ref] Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and..., Waters [/bib_ref] [bib_ref] Functional characterization of GPC-1 genes in hexaploid wheat, Avni [/bib_ref] , the lower grain Zn concentration in modern varieties can be explained. However, as stated above T. dicoccoides shows a high variation for micronutrients. Assuming that only selected T. dicoccoides genotypes were domesticated, there might be a chance that the chosen ones were low accumulators and this trait has been passed on to the domesticates. Regarding a negative effect on yield, the results are contradictory. In a study with recombinant chromosome substitution lines of T. durum and T. dicoccoides, the functional GPC-B1 allele did not affect yield [bib_ref] Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations, Distelfeld [/bib_ref] , while in another study a functional allele in hexaploid wheat led to reduced grain weights due to accelerated senescence [bib_ref] Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and..., Waters [/bib_ref]. In conclusion, the wild GPC-B1 allele appears to have a differential effect on different genotypes. When introducing this allele into modern varieties, the genotype-specific yield responses need to be taken into account. It would be interesting to see if there is an underling geographical pattern indicating environmental factors that have shaped genetic variation enabling biofortification. The bioavailability of micronutrients in cereal grains is limited by phytate, which binds Fe and Zn in insoluble forms and thus makes them unavailable for humans. To date, there is a lack of studies reporting phytate data from wheat wild relatives, which should be addressed in future research along with micronutrient analyses. Overall, the wheat genepool is still poorly explored in terms of diploid species and the timopheevii lineage. These species need to be considered in future research projects. Some of the transport and accumulation pathways of micronutrients in wheat have already been identified. The underlying translocation mechanisms can be targeted in wheat wild relatives to identify mechanisms that can be used in the breeding of biofortified wheat. ## Gluten in wheat wild relatives Gluten is the main form of storage protein in the grain and determines the baking quality. Up to 90% of the protein in the seed are gluten. The gluten protein group includes two components, glutenins and gliadins, which differ in molecular weight and sulfur (S) content. Glutenins control dough strength and elasticity but make up only for 10% of the seed storage protein. These polymers are classified according to their molecular weight into low molecular weight glutenin subunit (LMW-GS) and high molecular weight glutenin subunit (HMW-GS). The latter is further subdivided into x and y HMW-GS. Gliadins are monomers comprising the S-rich prolamins α/β and γ gliadins and the S-poor ω-gliadins. Gliadins are responsible Wheat wild relatives harbor rare y-type high molecular weight glutenin subunits Glutenins are important for the baking quality of the flour and form the backbone of the gluten network. Glutenin content increases in the order of diploid < tetraploid < hexaploid wheats [bib_ref] Comparative study on gluten protein composition of ancient (einkorn, emmer and spelt)..., Geisslitz [/bib_ref]. The Glu1 loci on the long arm of chromosome 1 homologous encode two HMW-GS genes, the x-and y-type subunits [bib_ref] Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin,..., Payne [/bib_ref] [bib_ref] Wheat storage proteins: their genetics and their potential for manipulation by plant..., Payne [/bib_ref]. The former has a higher molecular weight than the latter. HMW-GS that originate from the D genome influence dough properties the most . However, y-type subunits are also important for dough quality, although the y-subunit of the A genome (1Ay) is usually not expressed in hexaploid bread wheat [bib_ref] Electrophoretic analysis of the highmolecular-weight glutenin subunits of Triticum monococcum, T. urartu,..., Waines [/bib_ref]. Due to the higher number in cysteine residues compared to the x-subunit, the y-subunit is a valuable source for increasing dough quality . The variation in HMW-GS decreased with increasing ploidy status during evolution [bib_ref] Genetic diversity of HMW glutenin subunits in diploid, tetraploid and hexaploid Triticum..., Xu [/bib_ref]. The diploid wheat species showed the highest variation in molecules encoding the 1Ay subunit . The diploid A genome donor of wheat T. urartu contained different 1Ay subunit variants [bib_ref] Genetic diversity of HMW glutenin subunits in diploid, tetraploid and hexaploid Triticum..., Xu [/bib_ref] [bib_ref] Characterization of a novel 1Ay gene and its expression protein in triticum..., Hu [/bib_ref]. However, not all 1Ay subunits were expressed [bib_ref] Genetic diversity of HMW glutenin subunits in diploid, tetraploid and hexaploid Triticum..., Xu [/bib_ref]. This pattern was especially found in T. urartu accessions from Turkey, Armenia, Iraq and Iran [bib_ref] Electrophoretic analysis of the highmolecular-weight glutenin subunits of Triticum monococcum, T. urartu,..., Waines [/bib_ref]. Yet, the variation for 1Ay subunits was characterized by a rare allelic distribution. Prediction of the secondary structure of the 1Ay subunit of T. urartu showed a potentially beneficial structure for flour processing quality [bib_ref] Characterization of a novel 1Ay gene and its expression protein in triticum..., Hu [/bib_ref]. T. boeoticum may be another source of 1Ay subunit introduction [bib_ref] Electrophoretic analysis of the highmolecular-weight glutenin subunits of Triticum monococcum, T. urartu,..., Waines [/bib_ref]. The 1Ay subunit encoded by this species was different from the expressed 1Ay subunit of T. urartu [bib_ref] Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions, Jiang [/bib_ref]. The 1Ay subunit of T. boeoticum had a higher molecular mass and was thus longer, which favors the formation of an enhanced gluten network due to interchain bonds [bib_ref] Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions, Jiang [/bib_ref]. Introgression of the Glu-A1 locus of T. boeoticum into bread wheat resulted in an improved dough quality [bib_ref] Introduction to bread wheat (Triticum aestivum L.) and assessment for bread-making quality..., Rogers [/bib_ref]. The domesticated einkorn T. monococcum contained functional 1Ay subunits, two of which were investigated for their molecular characterization [bib_ref] Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12..., Guo [/bib_ref]. One allelic variant showed an additional cysteine residue, which could improve dough quality [bib_ref] Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12..., Guo [/bib_ref]. The diversity of 1Ay subunits was higher in T. boeoticum and T. monococcum than in T. urartu . Thus, the diploid wheats contain useful diversity for enriching dough quality, which should be exploited. T. dicoccoides contained the highest variation in 1Ay subunits among the tetraploid wheat species ). An active 1Ay subunit in T. dicoccoides (GenBank accession: JF519636) resembled the pattern of amino acid sequences of the 1Ay gene found in T. urartu, but they differed in their predicted secondary protein structure [bib_ref] Identification of an active 1Ay gene from Triticum turgidum ssp. dicoccoides, Bi [/bib_ref]. However, in a phylogenic analysis, this 1Ay allele of T. dicoccoides clustered together with another T. dicoccoides 1Ay subunit (GenBank accession: KC545956). This in turn was introgressed into hexaploid wheat and positively associated with improved dough quality ). The former subunit allele (JF519636) and probably other active 1Ay alleles of T. dicoccoides thus harbor putative beneficial quality traits. The wild tetraploid counterpart T. araraticum in the GGAA lineage showed only three different 1Ay subunits . Two of these 1Ay subunits were similar to T. boeoticum, and three were similar to T. monococccum ). In T. timopheevii, only one 1Ay subunit was found that matched a subunit from T. monococcum [bib_ref] Isolation and characterization of five novel high molecular weight subunit of glutenin..., Wan [/bib_ref] [bib_ref] Allelic variation and distribution of HMW glutenin subunit 1Ay in Triticum species, Hu [/bib_ref]. This variant also occurred in T. zhukovskyi. The other variant of the hexaploid GGA u A u Am A m T. zhukovskyi taxon did not occur in one of its putative ancestors . The variant 1Ay8 subunit in T. monococcum was favorable for bread making quality [bib_ref] Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12..., Guo [/bib_ref]. T. araraticum contained the homologous 1By8 subunit ), which could make T. araraticum a potential source for quality improvement. Wheat wild relatives show valuable y-type HMW-GS that promise improved dough quality. In particular, all diploid ancestors harbor favorable variants. The less explored GGAA lineage and especially T. zhukovskyi may be a valuable source for y-type subunits due to its kinship with both diploid A-genome wheat species, T. urartu and T. monococcum. This is also an advantage over the BBAA and BBAADD wheat species. As the diversity of the active 1Ay subunits decreased during wheat evolution and domestication history, it is of great importance to conserve the wild relatives. ## Alpha-gliadins in wild wheat-a safe option for celiac disease patients? The monomeric gliadins comprise a large gene family with high diversity due to their long evolutionary history. The Gli locus comprises genes that encode for gliadins. ω and γ gliadins are encoded by the Gli1 locus on chromosome 1 homologous. The short arm of chromosome 6 homologues contains the Gli2 locus, which encodes α/β gliadins . In addition to its quality-determining properties, gluten has a negative effect on patients with celiac disease (CD). The primary trigger for CD are α-gliadins, whereas the other gliadin groups appear to be less harmful but not entirely safe for some CD patients (Biesiekierski 2017). In the small intestine, gluten is digested (deamidated) and epitopes are formed during this process. These epitopes confer a T cell response that is activated by HLA-DQ2 and -DQ8 (HLA = Human leukocyte antigen) and leads to an autoimmune response (reviewed in: Biesiekierski 2017). Alpha-gliadins containing the 33mer peptide are most likely to cause CD. The 33mer peptide comprises the epitopes DQ2.5-glia-α1a and b, DQ2.5-glia-α2 and DQ2.5-glia-α3. The immunodominant 33mer fragment originates from the D genome [bib_ref] Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease..., Schaart [/bib_ref]. Therefore, diploid and tetraploid wheats might confer a less toxic immune response. Domesticated einkorn T. monococcum contained more gliadin and thus more gluten than common wheat. This observation was consistent across different locations, leading to the assumption that the higher gluten and gliadin are a characteristic of einkorn wheat [bib_ref] Comparative study on gluten protein composition of ancient (einkorn, emmer and spelt)..., Geisslitz [/bib_ref]. It is noteworthy that T. boeoticum, the wild ancestor of T. monococcum, showed higher gliadin content compared to its domesticate [bib_ref] Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae:..., Ozuna [/bib_ref]. However, at the molecular level, T. urartu and T. monococcum lacked some of the toxic epitopes and thus the 33mer peptide [bib_ref] Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for..., Molberg [/bib_ref] [bib_ref] Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins..., Salentijn [/bib_ref] [bib_ref] Genome-, transcriptome-and proteome-wide analyses of the gliadin gene families in triticum urartu, Zhang [/bib_ref] [bib_ref] Alpha-gliadin genes from the A, B, and D genomes of wheat contain..., Van Den Broeck [/bib_ref] [bib_ref] Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae:..., Ozuna [/bib_ref]. [bib_ref] Natural variation in toxicity of wheat: potential for selection of nontoxic varieties..., Spaenij-Dekking [/bib_ref] also found less alpha-gliadins in diploid A genomes. A comparative toxicity study suggested that gliadin of T. monococcum would be safer for CD patients than gliadin from T. aestivum [bib_ref] Lack of intestinal mucosal toxicity of Triticum monococcum in celiac disease patients, Pizzuti [/bib_ref]. However, T. monococcum harbors many different, putatively immunogenic and toxic epitopes; thus, it cannot be considered a CDsafe food only because of the absence of a 33mer peptide [bib_ref] A catalogue of Triticum monococcum genes encoding toxic and immunogenic peptides for..., Vaccino [/bib_ref] [bib_ref] Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide..., Ozuna [/bib_ref]. The same is true for T. urartu, which lacks the DQ2.5-glia-α2 epitope, but contains DQ2.5-glia-α1a and DQ2.5-glia-α3 CD-triggering epitopes [bib_ref] Genome-, transcriptome-and proteome-wide analyses of the gliadin gene families in triticum urartu, Zhang [/bib_ref]. This implies that regarding the quantity and molecular composition of gluten, einkorn is not a safe option for CD patients. Compared to einkorn, emmer wheat contained less gliadin and gluten, but more than hexaploid bread wheat [bib_ref] Comparative study on gluten protein composition of ancient (einkorn, emmer and spelt)..., Geisslitz [/bib_ref]. The potential B/G genome donor Ae. speltoides also lacked the 33mer peptide [bib_ref] Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for..., Molberg [/bib_ref] [bib_ref] Quantitation of the immunodominant 33-mer peptide from α-gliadin in wheat flours by..., Schalk [/bib_ref] , and conclusively, some of the T. dicoccon and T. durum samples showed the same phenotype [bib_ref] Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for..., Molberg [/bib_ref]. The B genome in tetraploid and hexaploid wheat showed the lowest expression level compared to Gli A2 and Gli D2 [bib_ref] Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins..., Salentijn [/bib_ref]. Further, it harbored the lowest amount of toxic epitopes [bib_ref] Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide..., Ozuna [/bib_ref]. Indeed, Ae. speltoides, Ae. longissima and Ae. searsii showed no abundance of any of the canonical epitopes. However, genes encoding for α-gliadins in T. dicoccoides encode toxic epitopes and thus constitute a threat for CD patients [bib_ref] Molecular characterization of alpha-gliadin genes from wild emmer wheat (Triticum dicoccoides), Qi [/bib_ref] [bib_ref] Alpha-gliadin genes from the A, B, and D genomes of wheat contain..., Van Den Broeck [/bib_ref] [bib_ref] Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae:..., Ozuna [/bib_ref]. However, the CD-triggering α-gliadin epitopes are most likely retrieved from the A genome [bib_ref] Alpha-gliadin genes from the A, B, and D genomes of wheat contain..., Van Den Broeck [/bib_ref]. Alpha-gliadins from the A genome in T. dicoccoides contained the T cell epitopes DQ2.5-glia-α1a and DQ2.5-glia-α3, which resembles the pattern in T. urartu [bib_ref] Rapid evolution of α-gliadin gene family revealed by analyzing Gli-2 locus regions..., Huo [/bib_ref] [bib_ref] Genome-, transcriptome-and proteome-wide analyses of the gliadin gene families in triticum urartu, Zhang [/bib_ref]. In summary, the B genome of tetraploid wheats can be considered the least toxic, but due to the presence of the A genome, tetraploid wheats remain unsuitable for CD patients. However, the content of toxic epitopes varied in tetraploid wheat and decreased as domestication progressed [bib_ref] Alpha-gliadin genes from the A, B, and D genomes of wheat contain..., Van Den Broeck [/bib_ref] [bib_ref] Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae:..., Ozuna [/bib_ref]. Thus, domesticated species seem to be more suitable for breeding wheat with fewer CD-triggering epitopes. It has to be kept in mind that this approach will generally only help a part of the CD patients, as the variability of toxic epitopes is high and the response to these depends on the individual. Ae. tauschii formed all of the three harmful epitopes. The DQ2.5-glia-α2 and DQ2.5-glia-α1b epitopes were exclusively assigned to the D genome and were only found in hexaploid wheat, suggesting that this epitope was inherited via the D genome donor [bib_ref] Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae:..., Ozuna [/bib_ref]. Ae. tauschii showed a high diversity for unique α-gliadin peptide variants, but only accessions from the potential geographic origin of hexaploid wheat hybridization (south-west Caspian Sea) contained the toxic 33mer peptide. On the one hand, this suggests that the CD-triggering epitope in bread wheat originates from this geographic region, but on the other hand, it also underlines that there are many Ae. tauschii accessions which might contain less to no toxic peptide variants [bib_ref] Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease..., Schaart [/bib_ref]. The 33mer peptide is the main trigger of CD; however, it seems that even partial epitopes can already cause a reaction in CD patients. Regarding the abundance of canonical epitopes, the D genome donor seems to show the highest profusion, followed by BAD and the A genome donor. The B genome is the least harmful in terms of toxic epitopes. It is still unclear whether the G genome has less toxic α-gliadins to offer, but the timopheevii lineage does not seem to be a promising resource for low α-gliadin epitopes, as the tetraploid T. araraticum and T. timopheevii potentially have the same A genome donor species as the emmer lineage. Moreover, T. zhukovskyi is probably even worse because the A genomes of T. urartu and T. monococcum are present. Nevertheless, one has to consider that the most toxic 33mer peptide comes from the D genome, which is absent in the timopheevii lineage. Therefore, it might be worthwhile to identify other epitopes there. ## Phenolic acids in the wheat genepool Phenolics occur in free, soluble conjugated and insoluble bound forms, with the latter form being the most abundant [bib_ref] Antioxidant activity of grains, Adom [/bib_ref]. The differently bound phenolic acids differ in their bioavailability. Ferulic acid is the major phenolic acid found in wheat [bib_ref] Phenolic compounds in wheat grain cultivars, Hernández [/bib_ref] , and it follows the pattern of the free, soluble conjugated and insoluble bound phenolics at a ratio of 0.1:1:100 [bib_ref] Antioxidant activity of grains, Adom [/bib_ref]. Wheat relatives have been investigated for their content in phenolic acids due to their antioxidant capacity. Data on the phenolics content of wild wheats are rare and contradictory. The wild diploid wheats T. urartu and T. boeoticum showed higher total polyphenol content (TPC) compared to the domesticated T. monococcum, T. dicoccon and T. aestivum, in which TPCs were almost the same [bib_ref] Phenolic acids and antioxidant activity of wild, feral and domesticated diploid wheats, Yilmaz [/bib_ref] , while T. durum grouped in between . The wild tetraploid emmer revealed the lowest phenolic content among domesticated emmer, T. durum and other tetraploid emmer landraces, thus implying inferiority of phenolic content of the wild ancestor of the tetraploid wheats [bib_ref] Genetic variation for phenolic acids concentration and composition in a tetraploid wheat..., Laddomada [/bib_ref]. Some data are available for the domesticated einkorn and emmer. Emmer showed a higher total phenolic content compared to einkorn, which was even lower than the total phenolic content of bread wheat [bib_ref] Phytochemical quantification and total antioxidant capacities of emmer (Triticum dicoccon Schrank) and..., Serpen [/bib_ref]. This pattern was also found in the ferulic acid content [bib_ref] Phytochemical quantification and total antioxidant capacities of emmer (Triticum dicoccon Schrank) and..., Serpen [/bib_ref]. The opposite was reported by [bib_ref] The effect of species and cultivation year on phenolic acids content in..., Barański [/bib_ref] , who measured significantly higher total phenolic acids in einkorn compared to emmer. However, this study only contained one einkorn line [bib_ref] The effect of species and cultivation year on phenolic acids content in..., Barański [/bib_ref]. Both studies revealed contradictory result compared to the equal phenolic content of emmer and einkorn reported by [bib_ref] Chemical composition of wild and feral diploid wheats and their bearing on..., Brandolini [/bib_ref]. A summary of studies analyzing T. monococcum, T. dicoccon, T. durum and T. aestivum with the same method and an overall comparison of studies with different methods were compiled by [bib_ref] Do "ancient" wheat species differ from modern bread wheat in their contents..., Shewry [/bib_ref]. In the former comparison, einkorn ranked last in the total phenolic acid and ferulic acid content and emmer ranked first, also showing a high variation. In their comparison of different studies, the authors found no distinct differences in the phenolics content across species due to high variability [bib_ref] Do "ancient" wheat species differ from modern bread wheat in their contents..., Shewry [/bib_ref]. In conclusion, no clear pattern can be identified from those studies. This might also be due to different analytical methods applied [bib_ref] Do "ancient" wheat species differ from modern bread wheat in their contents..., Shewry [/bib_ref]. Hence, there is no clear evidence that wild wheats possess valuable characteristics for the improvement in phenolic contents in wheat. ## The grain quality in an evolutionary context Evolution describes the gradual process of change and development in populations over time. Thereby, new characteristics or traits develop or disappear. The discussed grain quality characteristics micronutrients and gluten indicate an evolutionary pattern in their variation and accumulation. The main drivers for this phenomenon were a transition from wild to domesticated species and the change in ploidy levels. First of all, the variation for Fe/ Zn and 1Ay-glutenin subunits was higher in wild relatives of wheat compared to the domesticated species , [fig_ref] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies... [/fig_ref] [bib_ref] Zinc and iron concentrations in seeds of wild, primitive, and modern wheats, Cakmak [/bib_ref] [bib_ref] Triticum dicoccoides : An important genetic resource for increasing zinc and iron..., Cakmak [/bib_ref] [bib_ref] Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat..., Peleg [/bib_ref] [bib_ref] Allelic variation and distribution of HMW glutenin subunit 1Ay in Triticum species, Hu [/bib_ref]. This implies that during the transformation from wild to cultivated species, those traits were unintentionally altered and fixed, thus reducing the resulting diversity. However, the process of domestication also contributed to more stable grain quality traits, which is especially important for the baking quality. Besides the variation, the level of accumulation of Fe and Zn and α-gliadins also showed an evolutionary trend. The domesticated species harbored lower accumulation potential than the wild relatives. In the case of micronutrients, the gene GPC-B1, which turned non-functional during emmer domestication, underlies this phenomenon . High variation and accumulation potential for these grain constituents are thus features of wheat wild relatives that could be exploited in wheat improvement. In the case of phenolics, neither a clear pattern for changes in variation nor for an enrichment of phenolic content in wild and domesticated wheats was identified due to the lack of studies. However, it seems that there might be no evolutionary pattern for this secondary metabolite. Another evolutionary phenomenon that may have affected grain quality is allopolyploidization. The change in ploidy status caused different patterns for gliadin and glutenin during evolution: while an increase in ploidy reduced the gliadin content, the opposite was true for glutenin [bib_ref] Comparative study on gluten protein composition of ancient (einkorn, emmer and spelt)..., Geisslitz [/bib_ref]. Due to a lack of information in the scientific literature, the neglected timopheevii lineage should be analyzed regarding its grain quality. As with other wild species, the wild T. araraticum would be expected to exhibit high variations in Fe and Zn. Similar α-gliadin derived epitopes were found in T. urartu and T. dicoccoides and were both obtained from the A genome. Those epitopes would be likely to occur in the timopheevii lineage due to its ancestry with T. urartu, and furthermore, T. zhukovskyi might additionally contain the epitopes from T. monococcum. However, the toxic 33mer peptide is less likely to be represented in this lineage because of the missing hybridization with Ae. tauschii. Wild T. araraticum could also be a potential source for variation for glutenin subunits, 1 3 but higher glutenin concentration would be expected in the domesticated tetra-and hexaploid species due to their polyploidization status. These assumptions need scientific evidence, but if they hold true, they could support the concept that evolutionary history can help to identify beneficial species. # Conclusion The wild relatives of wheat harbor a large genetic diversity for certain grain quality traits such as minerals or gluten content. Additional important grain quality traits such as starch composition, fatty acid composition and non-starch polysaccharide concentration in wheat wild relatives have barely been investigated in the past. Thus, exploring the variation in these compounds in the wild relatives could be interesting topics for future studies. So far, the focus of most quality traits research has been on T. dicoccoides because of its straightforward use in bread wheat improvement. Identification of the underlying causative loci and polymorphism can be difficult in wild relatives due to the lack of reference genome sequences. However, ongoing advances in sequencing technology can simplify the development of reference genomes sequences for wheat wild relatives [bib_ref] Wild emmer genome architecture and diversity elucidate wheat evolution and domestication, Avni [/bib_ref] [bib_ref] Durum wheat genome highlights past domestication signatures and future improvement targets, Maccaferri [/bib_ref] [bib_ref] Genome sequence of the progenitor of wheat a subgenome Triticum urartu, Ling [/bib_ref] [bib_ref] Genome sequence of the progenitor of the wheat D genome Aegilops tauschii, Luo [/bib_ref] [bib_ref] Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia, Pont [/bib_ref]. In addition, the development of a wheat pan-genome sequence can be a valuable strategy in harnessing the genetic diversity of wheat wild relatives [bib_ref] Super-pangenome by integrating the wild side of a species for accelerated crop..., Khan [/bib_ref]. Furthermore, the advent of genome editing enables de novo domestication strategies for targeted use of crop wild relatives [bib_ref] De novo domestication: an alternative route toward new crops for the future, Fernie [/bib_ref] [bib_ref] Genome editing as a tool to achieve the crop ideotype and de..., Zsögön [/bib_ref] [bib_ref] De novo domestication of wild tomato using genome editing, Zsögön [/bib_ref] [bib_ref] De novo domestication towards new crops, Xie [/bib_ref]. However, ex situ conservation in genebanks constitutes an important strategy for safeguarding this unexplored diversity and also provides useful information/passport data of the accessions (https:// www. genes ys-pgr. org). In this context, the diploid taxa should receive more attention, due to their potential for enhancing grain micronutrient concentration as well as y-type HMW-GS. The same applies to the neglected GGAA lineage of wheat, in which quality traits remain to be investigated. In conclusion, further exploring the extended wheat gene pool harbors great potential for wheat diversification and quality improvement. Online Resource Map: https:// de.m. wikip edia. org/ wiki/ Datei:A_ large_ blank_ world_ map_ with_ oceans_ marked_ in_ blue. PNG. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. [fig] Figure 1: Origin and spread of domesticated einkorn wheat T. monococcum. Green dashed fields represent the Fertile Crescent; red circle is the domestication region of T. monococcum [/fig] [fig] Figure 2: Origin and spread of domesticated emmer and durum wheat. Green dashed fields are the Fertile Crescent; red circles are the domestication regions of T. dicoccon (Oliveira et al. 2020); solid arrows indicate dispersal routes of T. dicoccon (Martínez-Moreno et al. 2020; Badaeva et al. 2015); dashed arrows indicate dispersal route of T. durum(Moragues et al. 2007;Kabbaj et al. 2017;Martínez-Moreno et al. 2020); orange circle is the potential hybridization site of T. aestivum [/fig] [fig] Figure 3: Origin and spread of GGAA wheat. Green dashed fields are the Fertile Crescent; yellow circle indicates the center of diversity of T. araraticum [/fig] [fig] References 1: Cakmak et al. 2000; (2) Monasterio and Graham, 2000; (3)Cakmak et al.2004; (4) Chhuneja et al.2006; (5) Rawat et al. 2009; (6) Ozkan et al. 2007; [/fig] [fig] Figure 4: Distribution of mean Fe or Zn grain concentration measured in different studies for different species. The bold lines are the median of different species means. Dots represent individual mean values. Excel script from Weissgerber et al. (2015) [/fig] [fig] Figure 5: Regulatory role of the GPC-encoded NAC transcription factor for micronutrient translocation. Bold arrows indicate regulated transporters; dashed arrows show the transport of micronutrients. GPC = grain protein content gene; NAC = NAC transcription factor; IRT = iron-regulated transporter; ZRT = zinc-regulated transporter; HMA-like = Heavy Metal ATPase-like; NAAT = nicotianamine aminotransferase; PS = phytosiderophores for the viscosity of the dough(Wieser 2007; Biesiekierski 2017;Shewry 2019). The gluten composition in the grain is strongly dependent on the genotype(Wieser 2007). Thus, different Triticum species show diversity for different quality and health properties. [/fig]

Trends in socioeconomic inequalities in five major risk factors for cardiovascular disease in the Korean population: a cross-sectional study using data from the Korea National Health and Nutrition Examination Survey, 2001–2014 MenEducation(yr) ≥13 56. ## Men Education(yr) ≥13 56.6(56.6-56.7) 46.6(46.5-46.7) 42.0(41.9-42.1) 42.6(42.6-42.7) 41.9(41.9-42.0) 10-12 68.6(68. 2.8(2.8-2.8) 5.2(5.2-5.2) 6.1(6.0-6.1) 6.8(6.8-6.8) 6.1(6.1-6.2) Q4 (lowest) 6.6(6.6-6.7) 9.0(9.0-9.1) 9.0(9.0-9.0) 10.9(10.8-10.9) 10. 8.9(8.9-8.9) 10-12 9.4(9.4-9.5) 8.9(8.9-8.9) 9.5(9.5-9.5) 10. .9(7.9-8.0) 9.3 (9.2-9.3) 9.5(9.5-9.6) Q2 6.6(6.6-6.6) 8.6(8.6-8.6) 7.3(7.3-7.3) 8.1(8.1-8.2) Q3 9.0(9.0-9.0) 7.8(7.8-7.9) 8.6(8.6-8.7) 9.8(9.8-9.8) Q4 (lowest) 10.4(10.3-10.4) 11.4(11.3-11.4) 9.8(9.7-9.8) 12.1(12.1-12.2) SII 1.05 3.75 1.16 3.83 P for SII linear trend 0.576 Women Education(yr) ≥13 0.6(0.6-0.6) 2.8(2.8-2.8) 3.8(3.8-3.8) 3.9(3.8-3.9) 10-12 5.8(5.7-5.8) 5.2(5.1-5.2) 5.5(5.5-5.5) 5.6(5.6-5.7) 7-9 7.7(7.6-7.7) 5.3(5.3-5.4) 5.0(5.0-5.1) 7.5(7.4-7.5) ≤6 7.2(7.1-7.2) 9.2(9.1-9.2) 8.1(8.1-8.2) 7.3(7.3-7.4) SII 8.92 7.00 4.55 5.06 P for SII trend 0.048 Income Q1 (highest) 6.1(6.1-6.1) 3.9(3.9-4.0) 2.9(2.8-2.9) 4.3(4.3-4.3) Q2 5.1(5.1-5.1) 4.2(4.1-4.2) 4.9(4.9-5.0) 4.6(4.6-4.6) Q3 6.4(6.4-6.4) 6.7(6.7-6.7) 6.4(6.3-6.4) 5.1(5.0-5.1) Q4 (lowest) 4.4(4.1-4.4) 7.5(7.5-7.5) 6.9(6.8-6.9) 7.9(7.9-7.9) SII - 7.6(7.6-7.7) 8.9(8.8-9.0) 8.9(8.9-9.0) 12.2(12.0-12.4) SII -1.40 -0.28 -3.02 2.80 P for SII trend 0.378 Income Q1 (highest) 8.0(8.0-8.1) 9.9(9.9-9.9) 13.1(13.0-13.1) 13.6(13.6-13.7) Q2 4.8(4.8-4.9) 8.7(8.6-8.7) 8.9(8.9-8.9) 14.7(14.6-14.7) Q3 7.6(7.6-7.7) 8.8(8.7-8.8) 12.9(12.9-12.9) 10.9(10.9-11.0) Q4 (lowest) 8.9(8.9-9.0) 10. .7(9.7-9.8) 13.7(13.7-13.8) 11.0(11.0-11.0) 7-9 7.1(7.0-7.1) 10.3(10.3-10.4) 11.2(11.1-11.2) 9.8(9.7-9.8) ≤6 5.2(5.1-5.2) 11.9(11.8-11.9) 9.8(9.8-9.9) 10.4(10.3-10.4) SII -10.25 -1.89 -3.27 -3.20 P for SII trend 0.177 Income Q1 (highest) 8.0(7.9-8.0) 10.2(10.2-10.3) 12.4(12.4-12.5) 11.8(11.8-11.9) Q2 7.4(7.4-7.4) 9.2(9.2-9.3) 12.2(12.1-12.2) 9.8(9.8-9.8) Q3 5.6(5.5-5.6) 10.0(10.0-10.0) 11.7(11.7-11.8) 14.2(14.2-14.3) Q4 (lowest) 7.6(7.6-7.7) 10.9(10.9-10.9) 13.8(13.8-13.8) 11.7(11.6-11.7) SII -1.08 1.08 1.49 1.58 P for SII linear trend 0.535 Narrow CI's are due to small SE for prevalence by using weighted samples, which are as large as whole population, for direct standardization

A Mechanistic Review of β-Carotene, Lutein, and Zeaxanthin in Eye Health and Disease Carotenoids are natural lipid-soluble antioxidants abundantly found as colorful pigments in fruits and vegetables. At least 600 carotenoids occur naturally, although about 20 of them, including β-carotene, α-carotene, lycopene, lutein, zeaxanthin, meso-zeaxanthin, and cryptoxanthin, are detectable in the human blood. They have distinct physiological and pathophysiological functions ranging from fetal development to adult homeostasis. β-carotene is a precursor of vitamin A that essentially functions in many biological processes including vision. The human macula lutea and eye lens are rich in lutein, zeaxanthin, and meso-zeaxanthin, collectively known as macular xanthophylls, which help maintain eye health and prevent ophthalmic diseases. Ocular carotenoids absorb light from the visible region (400-500 nm wavelength), enabling them to protect the retina and lens from potential photochemical damage induced by light exposure. These natural antioxidants also aid in quenching free radicals produced by complex physiological reactions and, consequently, protect the eye from oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation. This review discusses the protective mechanisms of macular xanthophylls in preventing eye diseases such as cataract, age-related macular degeneration, and diabetic retinopathy. Moreover, some preclinical animal studies and some clinical trials are discussed briefly to understand carotenoid safety and efficacy. # Introduction The major causes of progressive and irreversible loss of vision include various ophthalmic diseases such as cataract, age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Initiation and progression of these disorders involve oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation . For instance, increased oxidative stress of retinal cells damages the mitochondrial DNA in diabetic retinopathy, one of the most deleterious eye-related complications of diabetes. Oxidative stress is also a significant contributor to the pathophysiology of age-related cataract, a leading cause of blindness globally. A growing body of evidence indicates that dietary antioxidants can prevent and treat many ophthalmic disorders associated with oxidative stress. Lutein, zeaxanthin, and meso-zeaxanthin (synthesized from lutein in the retina) are dipolar, terminally dihydroxylated carotenoids, also known as macular xanthophylls, and are obtained from dietary sources. Macula lutea of the eye, also known as the yellow spot, contains high concentrations of macular xanthophylls. The peak concentrations of lutein and zeaxanthin appear at the center of the fovea. Lutein and zeaxanthin are also found in the lens; however, β-carotene and lycopene have not been detected. macular xanthophylls has been proposed. Macular xanthophylls located transversely in the lipid bi-layer of the retinal membrane are able to prevent AMD and protect the retina against peroxidation and photo-damage by acting as antioxidants that quench free radicals and ROS. They also prevent blue light exposure to fovea's photoreceptors significantly . . Schematic diagram showing the mechanisms of action of carotenoids to prevent age-related macular degeneration (AMD). HR-LBP: human retinal lutein-binding protein; GSTP1: glutathione S-transferase Pi 1; R: free radical (symbolic representation).isolated and purified a membrane-associated xanthophyll-binding protein from human macula using ion-exchange chromatography and gel-exclusion chromatography. This protein is a Pi isoform of human glutathione S-transferase (GSTP1) to which zeaxanthin displayed the highest affinity. Uptake, metabolism, and stabilization of zeaxanthin in the retina were found to be mediated by this xanthophyll-binding protein. The HR-LBP, a membrane-associated human retinal lutein-binding protein, displayed a saturable and specific binding toward lutein. Once incorporated in the lipid bilayer, macular xanthophylls help quench singlet oxygen and other free radicals and thus prevent lipid peroxidation in the retina. Carotenoids also protect against oxidative damage by repairing α-tocopherol and acting synergistically with vitamin C. illustrates the mechanisms of action of ocular carotenoids to prevent AMD. ## Cataracts A cataract is a visualization problem in which the lens develops opacity, and age-related cataract is a leading cause of blindness. Depending on the morphology, cataract is classified into different types. The outer section of the tissue becomes opaque in cortical cataracts, the inner core in nuclear cataracts, and the superficial region below the capsule on the posterior side in posterior subcapsular cataracts. In western countries, cataract surgery is most frequently done in people aged 65 years or older. This is one of the most common surgical procedures among the general population, and the prevalence is increasing each year. In the United States, 3.38 million cataract surgeries were performed in 2017. Although cataract is mainly an age-related phenomenon, socioeconomic and lifestyle factors (smoking, diet, intake of nutrients, alcohol consumption, etc.) also influence cataract initiation and progression. The main constituents of an eye lens are crystallins (90%), and cytoskeletal and membrane proteins. Crystallins have a high refractive index and form a complex protein solution in the cytoplasm of lens fibers, conferring transparency. With age, this protein slowly leaves the soluble phase. Subsequently, disulfide bond formation and non-enzymatic glycation alter attractive forces between lens proteins.observed aspartic acid racemization during aging and cataract formation on a D/L enantiomeric analysis of control human lenses and cataracts. The insoluble fraction of D-aspartic acid becomes less abundant in cataractous lenses. Thus, crystallins may undergo various post-translational modifications such as oxidation, glycation, proteolysis, transamidation, carbamylation, and phosphorylation. These changes result in aggregation of proteins, disruption of healthy lens cell structure, and opacification. Ocular oxidative stress may result from an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense mechanisms and subsequently initiate lens opacification. ROS, such as hydrogen peroxide, superoxide, and hydroxyl radicals, negatively modifies the lens, whereas antioxidants, including glutathione (GSH), ascorbate, and catalase, rescue the lens proteins against ROS. Hydrogen peroxide, the primary oxidant in the pathogenesis of cataract, is eliminated by catalase and glutathione through enzymatic reactions. A decreased level of reduced glutathione in older lenses' nucleus promotes cataract formation. An imbalance in redox reactions can also initiate lipid peroxidation, promoting cataractogenesis.mentioned that the massive oxidation of thiol to protein and mixed disulfides, cysteic acid, and methionine sulfoxide and cataract-extensive methionine sulfoxide formation are common in older lens. In the nucleus of nuclear cataracts, covalently linked disulfide bonds containing polypeptides and in cortical cataracts, high molecular weight disulfide-linked aggregates were found. Thus, oxidation of crucial sulfhydryl groups of enzymes and membrane proteins and the peroxidation of lenticular plasma membrane lipids also contribute to cataract pathogenesis. Carotenoids' roles as antioxidants are known for many decades. β-carotene was found to markedly inhibit lipid peroxidation induced by xanthine oxidase in a pioneering study by and Fridovich. Chemical antioxidants (e.g., α-tocopherol, β-carotene, ascorbate, and GSH) and structural antioxidants (e.g., cholesterol and membrane protein) are implicated in preventing oxidative damage of the ocular tissues.reviewed antioxidants' protective effects in cataract and macular degeneration and found that animal studies invariably advocated in favor of dietary antioxidants, although results from epidemiological analyses were inconclusive. The mechanisms of preventive functions of carotenoids in cataract formation are shown in. Human lens contains lutein and zeaxanthin but not β-carotene. It has been suggested that antioxidants lutein and zeaxanthin are delivered continuously from the body pool to the epithelial/cortical layer of the lens, where they scavenge ROS by up-regulating GSH, catalase and SOD activities.reported that lutein and zeaxanthin could reduce the risk for senile cataract by protecting lens protein, lipid, and DNA from oxidative damage. They incubated human lens epithelial cells with or without 5 µM lutein, zeaxanthin, or α-tocopherol for 48 h. Then the cells were exposed to 100 µM H 2 O 2 for 1 h to induce oxidative stress. By using a battery of in vitro analyses, the authors observed that the levels of H 2 O 2 -induced protein carbonyl, MDA, and DNA damage were significantly reduced by lutein and zeaxanthin. Interestingly, cataract patients exhibited increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of MDA was significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were substantially lower in age-related cataract patients compared to healthy volunteers. Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 22 exhibited increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of MDA was significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were substantially lower in age-related cataract patients compared to healthy volunteers. ## Diabetic retinopathy Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy. Diabetic retinopathy is prevalent in people with Type 1 and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been identified as a risk factor for the progression of diabetic retinopathy. Carotenoids enhance insulin sensitivity and have a protective effect against diabetes-related infectious diseases. In diabetes, the high glucose level present in the microvasculature of the retina compromises the electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the retina. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature, including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation, and advanced glycation end-product formation. Rat retinal endothelial cells exposed to high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway and increased apoptosis. HG was also found to increase mitochondrial fragmentation and proapoptotic cytochrome c levels in vascular cells of rat retinal capillaries. Increased oxidative ## Diabetic retinopathy Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy. Diabetic retinopathy is prevalent in people with Type 1 and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been identified as a risk factor for the progression of diabetic retinopathy. Carotenoids enhance insulin sensitivity and have a protective effect against diabetes-related infectious diseases. In diabetes, the high glucose level present in the microvasculature of the retina compromises the electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the retina. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature, including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation, and advanced glycation end-product formation. Rat retinal endothelial cells exposed to high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway and increased apoptosis. HG was also found to increase mitochondrial fragmentation and pro-apoptotic cytochrome c levels in vascular cells of rat retinal capillaries. Increased oxidative stress, elevated oxidatively modified DNA, and up-regulated nitrosylated proteins ensue an impairment in antioxidant defense enzymes, which eventually leads to increased retinal capillary cell apoptosis. Further, mitochondrial metabolism generates ROS, such as superoxides and hydrogen peroxide, that can damage proteins, lipids, and DNA. The damage of proteins can be compensated because of continuous biosynthesis; however, DNA damage can be devastating if a fixed mutation occurs. If reactive oxygen species damage a portion of a single DNA strand (e.g., the addition of 8-oxo-2 -hydroxyguanine in DNA strand) and DNA polymerases copy that damaged templates during replication, then, this error becomes permanent. DNA double-strand can also be affected and broken by free radicals. This breakdown is usually repaired by ligating nonhomologous DNA ends, an error-prone repair system. In an in vitro study, Santos, Tewari and Kowluru, (2012) observed that the damage caused by ROS was compensated by increased mitochondrial DNA biosynthesis and repair system in the early stages of diabetes (15 days to 2 months). At a stable diabetic condition (at 6 months of diabetes) with constant production of high ROS, mitochondrial DNA and electron transport chain (ETC) were damaged because repair/replication machinery became subnormal and mitochondrial DNA copy number was significantly decreased. An increase in apoptosis was also observed in the above study. Compromised DNA repair machinery, decreased gene expressions of mitochondrial-encoded proteins, and increased mtDNA damage were observed at high glucose exposure of retinal endothelial cells.observed a higher amount of advanced glycation end-products (non-enzymatic binding of glucose to free amino groups of an amino acid) in patients with retinopathy. In hyperglycemic conditions, a high glucose level causes overproduction of a glycolytic metabolite glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate can easily be converted into 1, 3 Diphosphoglycerate by converting nicotinamide adenine dinucleotide (NAD+) into to its reduced form (NADH) when ROS inhibits the overproduction of glyceraldehyde-3-phosphate dehydrogenase (GADPH). NADH facilitates the protein kinase C (PKC) pathway and the AGE pathway. Advanced glycation end-products (AGEs) (glucosepane and methylglyoxal hydroimidazolone) were significantly associated with the progression of retinopathy. Antioxidants such as ascorbate, tocopherol, and carotenoids protect ocular oxidative damage. Carotenoids can quench free radicals, scavenge reactive oxygen species, modulate gene expression, reduce inflammation, and prevent diabetes-related microvascular complications, including diabetic retinopathy, nephropathy, and neuropathy. Macular pigment (MP), including lutein, zeaxanthin and mesozeaxanthin also contributes to the protection of the retinal tissue by conferring potent antioxidant and anti-inflammatory effects in diabetes. It has been demonstrated that patients with type 2 diabetes have a lower level of MP as compared to healthy controls. Lutein supplementation is known to prevent oxidative damage in the retina. In a mouse model of early diabetic retinopathy, long-term lutein administration attenuated inflammation, and vascular damage of the retina. Intriguingly, short-term lutein treatment also down-regulated reactive oxygen species and up-regulated superoxide dismutase (SOD), attenuating inflammation and protecting the photo-stressed retina from oxidative damage. Several signaling pathways, including PKC, vascular endothelial growth factor (VEGF), nuclear factor erythroid 2-related factor 2 (Nrf2), and Rho/Rho-associated coiled-coil containing protein kinase (Rho/ROCK), have been implicated in carotenoid-mediated protection of the retina in diabetic retinopathy. An in vitro study showed that co-administration of lutein and zeaxanthin attenuated VEGF-induced oxidative stress in the retinal endothelium. Lutein was found to modulate the SIRT1 signaling and inhibit premature senescence in retinal pigment epithelium cells. Recent studies indicate that carotenoids could exert therapeutic benefits in diabetic retinopathy through multiple cellular and molecular pathways. The mechanisms of action of carotenoids to prevent diabetic retinopathy discussed above are sketched in. ## Safety of carotenoids Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation at different doses and duration in experimental animals. summarizes some of these outcomes. ## Safety of carotenoids Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation at different doses and duration in experimental animals. summarizes some of these outcomes. ## Clinical trials Clinical trials are the final step assessments of any drug before it is approved for regular human application. Twenty-siximportant clinical trials are summarized into understand the efficacy of carotenoids as prophylactic and therapeutic uses in eye diseases. Serum level increased linearly with increased dose. Group 3 showed the highest ratio of MPOD change, which was statistically significant (p = 0.021). Preclinical study results listed in have established the safety profile of several carotenoids. Results from different clinical trials listed inconfirm that an increased serum level of lutein was correlated with enhanced visual acuity. An increase in macular pigment optical density (MPOD) was seen with lutein and zeaxanthin supplementation. Several studies reported that lutein and zeaxanthin administration was associated with a reduced risk of cataract. Nonetheless, some studies did not find statistically significant effects of lutein and zeaxanthin on prevention of eye diseases or enhancement of macular pigments. In a study by, lutein and zeaxanthin were found in the lens, but β-carotene and lycopene were not detected. Among six clinical trials mentioned in, which examined the effects of β-carotene on cataract, five found no significant impact, and one showed a small reduction in the progression of age-related cataract. A study found that smokers, people with high BMI, a history of hypertension, diabetes, and high cholesterol developed cataracts even after taking antioxidants. These studies suggest that β-carotene is not adequate for cataract prevention as the lens does not contain any β-carotene. On the contrary, in vivo studies on animals showed that lutein is safe even at a very high dose, and the LD 50 of lutein exceeded 10,000 mg/kg body weight. Eight in vivo studies were mentioned in this review , and none of these studies observed any significant adverse effect or toxicity.suggested a daily intake of 3 mg/kg/day meso-zeaxanthin for human. Intake of up to 20 mg/day for lutein was found to be safe for humans. # Conclusions In this review, we summarized the detrimental effects of ocular oxidative stress generated from the continuous exposure of ultraviolet and blue lights. Then we discussed the protective roles of carotenoids, namely β-carotene, lutein, and zeaxanthin, against three distinct eye diseases, highlighting the outcomes from the clinical trials. A considerable number of studies including preclinical and clinical trials demonstrated that β-carotene, lutein, and zeaxanthin can prevent the progression of eye diseases, mainly by quenching free radicals and preventing oxidative damage to the retina. According to study outcomes, it is obvious that β-carotene, lutein, and zeaxanthin can efficiently attenuate oxidative stress in vivo and confer protection to the eye. The biological functions of different carotenoids in human are established. As the human body cannot synthesize this important class of molecules, they must be supplied as dietary intake or food/pharmaceutical supplement. Thus, the optimum levels of cellular concentrations of β-carotene, lutein, and zeaxanthin in eye tissue may help maintain eye health. It is noteworthy that carotenoids, particularly MP, can be assessed noninvasively in retina; such assessment may be useful to determine the average dietary intake of lutein and zeaxanthin to meet the regular need of these molecules. It has been shown that MP attenuates oxidative stress and slows down the progression of apoptosis, mitochondrial dysfunction, and inflammation in diabetes, which can be improved by increasing dietary supplementation of lutein and zeaxanthin. A long-term cohort study byfound a remarkable 40% reduced risk of advanced AMD progression for predicted plasma lutein/zeaxanthin scores. Nevertheless, carotenoids show a high degree of variability in bioavailability, which poses a challenge for finding suitable forms (as foods, supplements, or medicines) that can be administered to the patients with AMD. Gastrointestinal absorption and subsequent distribution to ocular tissues are influenced by dietary factors, formulations, gender, age, disease states, and individual genetic variations. A recent study found a significantly higher absorption of zeaxanthin and meso-zeaxanthin from a diacetate micromicelle preparation than free carotenoid preparations. Intriguingly, a nano-formulation of lutein-poly-(lactic-co-glycolic acid) (PLGA)-phospholipid (PL) showed a significantly elevated level of lutein in plasma when administered at a lower dose in mice. Novel drug delivery systems and formulations thus could further be exploited to achieve favorable pharmacokinetic and pharmacodynamic profiles of macular xanthophylls in humans. In addition, long-term clinical trials with large numbers of populations may be undertaken to confirm the effects of these molecules. Future studies will substantiate the therapeutic potentials of different β-carotenoids.

Venom immunotherapy: safety and tolerability of the build‐up phase with depot versus aqueous preparations ## R e s e a r c h l e t t e r venom immunotherapy: safety and tolerability of the build-up phase with depot versus aqueous preparations To the Editor, Insect stings can cause an allergic reaction ranging from large local reaction (LLR) to systemic sting reactions (SSRs). Venom immunotherapy (VIT) is the only effective treatment in preventing systemic reactions at the subsequent field sting. VIT includes the so-called build-up phase and the maintenance phase, according to European and Italian guidelines. [bib_ref] EAACI guidelines on allergen immunotherapy: hymenoptera venom allergy, Sturm [/bib_ref] [bib_ref] Hymenoptera venom allergy: management of children and adults in clinical practice, Bilò [/bib_ref] An EAACI multicentre study revealed an incidence of 20% side-effects to VIT, the majority of which occurred in the build-up phase. [bib_ref] Side-effects of insect venom immunotherapy: results from an EAACI multicenter study, Mosbech [/bib_ref] In another European multicentre study, the risk of systemic reaction during build-up phase was 8.4%. [bib_ref] European Academy of Allergy and Clinical Immunology Interest Group. Predictors of side..., Ruëff [/bib_ref] In Europe, available Hymenoptera venom preparations used to perform VIT are divided into aqueous and depot preparations, which can be purified or not. [bib_ref] EAACI guidelines on allergen immunotherapy: hymenoptera venom allergy, Sturm [/bib_ref] In most European countries, VIT is still performed with aqueous preparations, especially during VIT induction phase with different protocols (slower or faster), while the maintenance phase can be pursued with depot preparations. [bib_ref] Higher frequency of early local side effects with aqueous versus depot immunotherapy..., Cadario [/bib_ref] Depot preparations are generally associated with fewer local side-effects than aqueous preparations during VIT maintenance phase. The slower build-up phase with depot preparations and the slow release from the injection site of allergen adsorbed on to aluminium hydroxide or L-tyrosine are considered as an advantage. [bib_ref] Higher frequency of early local side effects with aqueous versus depot immunotherapy..., Cadario [/bib_ref] [bib_ref] Safety of hymenoptera venom immunotherapy: a systematic review, Incorvaia [/bib_ref] At the moment only few studies, based on a limited population size, provide definite evidence on build-up phase side-effects using depot versus aqueous venom preparations. Instead, there is little doubt about the efficacy and the lower side-effect rate using depot preparations during VIT maintenance phase. [bib_ref] EAACI guidelines on allergen immunotherapy: hymenoptera venom allergy, Sturm [/bib_ref] [bib_ref] Higher frequency of early local side effects with aqueous versus depot immunotherapy..., Cadario [/bib_ref] The aim of this study is to retrospectively evaluate the safety of VIT build-up phase comparing commercially available purified depot and aqueous venom preparations. Between January 2010 and July 2020, patients with a clinical history of hypersensitivity to Hymenoptera venom, consecutively administered VIT according to EAACI Guidelines and Italian Consensus criteria, 1,2 were retrospectively enrolled in the study as notified to the Ethical Committee. After appropriate informed consent, patients decided to be admitted to a 3-week protocol with purified aqueous preparation or to a 6-week protocol with puri- No antihistamine or Omalizumab premedication were used, as the first could mask a mild reaction, while the second is considered offlabel in Italy during VIT. Venom purified depot preparations were: Alutard SQ adsorbed onto aluminium hydroxide (ALK-Abellò) and L-tyrosine-adsorbed preparation (Anallergo). Venom purified aqueous preparations were: Aquagen SQ (ALK-Abellò) and Anallergo (Florence, Italy). Systemic and local side-effects were recorded in the outpatient regimen procedure. LLRs and SRs were treated, depending on the severity. When no reactions occurred, the observation time after the last administered dose was 3 h. All patients were instructed to report any delayed reactions to the Allergy Unit, and they were interviewed during the next visit about any reaction or discomfort occurred within 24 h after VIT. Data were stored in a Microsoft Access database. We compared two groups (patients receiving build-up phase with depot vs. aqueous preparations) using chi-squared or Wilcoxon rank-sum test, for categorical and quantitative variables respectively. We fitted multivariable Poisson regression models with robust variance to calculate risk ratios (RR) of adverse reactions and 95% confidence intervals (CI) according to preparation type (depot vs. aqueous) adjusted for selected potential confounders, including gender, age class, venom type and Mueller grade. In a sensitivity analysis, we fitted a model using continuous age (in decades). Analyses were performed with Stata 17 (StataCorp. 2021). We consecutively enrolled 444 adult venom allergic patients (age range 18-86 years). Among these, we excluded: three patients treated with two venom preparations (wasp and honeybee), eight patients suffering from systemic mastocytosis (due to likely higher reaction risk) and 15 patients who underwent a rush induction protocol (as faster protocols may be associated with more frequent adverse events). 1 Statistical analyses were performed on 418 patients: 258 (61.7%) and 160 (38.3%) were respectively treated with purified depot or purified aqueous preparations ( ## Key messages - Depot preparation during VIT induction reduced risk of LLRs by 85% compared to aqueous preparation. - Preparation type (depot or aqueous) did not influence risk of systemic reaction. - Risk of adverse reaction linearly increased with age by 27% every decade. In conclusion, notwithstanding these limitations, the adjusted relative risk of adverse reactions during build-up phase was 6-7 times lower with depot than aqueous preparations. Based on these results, our study supports the safety and tolerability of depot preparations used for the VIT build-up phase. Future studies are needed to confirm and better characterize our findings. ## Ta b l e 1 demographic data and clinical characteristics of the 418 patients under investigation ## Ack n owled g m ent ## Co n fli c t o f i nte r e s t The authors declare they have no conflicts of interests. ## Data ava i l a b i l i t y s tat e m e n t The data that support the findings of this study are available from the corresponding author upon reasonable request. [table] Table 1: Among patients submitted to VIT with depot formulation, there were more males than females (70.1% vs. 29.9%). A similar This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd. distribution was observed among patients treated with aqueous preparations (75.6% vs. 24.4%). Patients treated with depot preparations were younger (average 50.1 years, range: 18.7-78.3) than those with aqueous preparations (54.5 years, range: 18.0-85.9; p = .005). Hymenoptera venom types were equally distributed: wasp VIT was the most frequently performed both in patients treated with depot (87.2%, 225 of 258) and aqueous preparations (88.8%, 142 of 160; p = .64); honeybee VIT was less frequently performed both in patients treated with depot (12.8%, 33/258) and aqueous preparations (11.2%, 18/160). The overall cumulative incidence of reactions during induction phase was 9.1% (38/418; 95% CI: 6.7-12.3%). In patients treated with depot preparations 5 LLRs and 7 SRs occurred, while among those treated with aqueous preparations 20 LLRs and 6 SRs occurred. The risk of SRs was similar in both populations: 2.7% with depot preparations and 3.7% with aqueous preparations (p = .55). Considering LLR, females had a slightly elevated risk compared to males (Table 2). LLR occurred only among subjects aged 40 years or more. The multivariable Poisson model with continuous age yielded a RR of 1.27 (95% CI: 1.01-1.60), that is, a 27% increase per decade of age (95% CI: 1-60%). The Mueller grade pre-VIT had low influence on LLR during the build-up phase. Patients treated with honeybee venom had a slightly reduced adjusted risk, but with a wide CI. The relative risk of LLR was much lower (RR 0.16, 95% CI: 0.06-0.41, p < .001) in those treated with depot preparations (1.9%) compared to those [/table]

COVID-19 what have we learned? The rise of social machines and connected devices in pandemic management following the concepts of predictive, preventive and personalized medicine Objectives Review, compare and critically assess digital technology responses to the COVID-19 pandemic around the world. The specific point of interest in this research is on predictive, preventive and personalized interoperable digital healthcare solutions. This point is supported by failures from the past, where the separate design of digital health solutions has led to lack of interoperability. Hence, this review paper investigates the integration of predictive, preventive and personalized interoperable digital healthcare systems. The second point of interest is the use of new mass surveillance technologies to feed personal data from health professionals to governments, without any comprehensive studies that determine if such new technologies and data policies would address the pandemic crisis. Method This is a review paper. Two approaches were used: A comprehensive bibliographic review with R statistical methods of the COVID-19 pandemic in PubMed literature and Web of Science Core Collection, supported with Google Scholar search. In addition, a case study review of emerging new approaches in different regions, using medical literature, academic literature, news articles and other reliable data sources. Results Most countries' digital responses involve big data analytics, integration of national health insurance databases, tracing travel history from individual's location databases, code scanning and individual's online reporting. Public responses of mistrust about privacy data misuse differ across countries, depending on the chosen public communication strategy. We propose predictive, preventive and personalized solutions for pandemic management, based on social machines and connected devices. Solutions The proposed predictive, preventive and personalized solutions are based on the integration of IoT data, wearable device data, mobile apps data and individual data inputs from registered users, operating as a social machine with strong security and privacy protocols. We present solutions that would enable much greater speed in future responses. These solutions are enabled by the social aspect of human-computer interactions (social machines) and the increased connectivity of humans and devices (Internet of Things). Conclusion Inadequate data for risk assessment on speed and urgency of COVID-19, combined with increased globalization of human society, led to the rapid spread of COVID-19. Despite an abundance of digital methods that could be used in slowing or stopping COVID-19 and future pandemics, the world remains unprepared, and lessons have not been learned from previous cases of pandemics. We present a summary of predictive, preventive and personalized digital methods that could be deployed fast to help with the COVID-19 and future pandemics. # Introduction With the rise of COVID-19, most western governments seem determined to design apps and surveillance mechanisms as a means of response. During the first wave of the COVID-19 pandemic, these proved successful in some countries including Taiwan, South Korea and China. Western countries are developing similar monitoring and surveillance approaches, hoping to replicate that success and prevent significant damage from rising risks of a second wave. With monitoring as the main drive, we could be limiting the potential for pandemic management. Designing separate systems in isolation could lead to the realization that such systems are coupled by default. But by being designed in isolation, such systems become proprietary and not interoperable with similar systems built by others. This can also result in the repeat of past cybersecurity mistakes. We investigate interoperability in predictive, preventive and personalized medical system design, which could become the topic of concern when upgrading the same systems we are building today, for pandemic management at the latter stages of this outbreak or future outbreaks. ## The case of covid-19 COVID-19 was declared as an outbreak in January 2020 by the World Health Organization (WHO). Within 6 weeks, it became a pandemic, and by mid-March 2020, the COVID-19 pandemic had generated 24 times more cases than the severe acute respiratory syndrome (SARS) outbreak. One of the differences is that the amount of digital data produced in the time period prior to and during SARS in 2003 is produced within minutes today in 2020. Big data has enabled geospatial mapping of COVID-19 for epidemic transition analysis, but challenges remain in 'data aggregation, knowledge discovery, and dynamic expression'. There is also an opinion that many countries did not learn from the previous two coronavirus pandemics (Middle East respiratory syndrome (MERS) and SARS), resulting in an inadequate risk assessment of the urgency and the speed of the pandemic [bib_ref] The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest..., Peeri [/bib_ref]. Taiwan on the other hand has learned from the SARS pandemic and has developed a fast response health mechanism that recognized the crisis and activated emergency measures to contain the outbreak [bib_ref] Response to COVID-19 in Taiwan: big data analytics, new technology, and proactive..., Wang [/bib_ref]. ## Digital solutions for the covid-19 outbreak This review paper is looking for digital solutions that enhance the speed of response during pandemics. Fast information flow is needed for understanding the dynamics and development of an epidemic and to aid prevention, control, and decision-making and for informing actions. This research studies interoperability of digital health solutions and the benefits from integration of these systems. A lack of data in the initial stages of the COVID-19 outbreak led to its initial rapid spread across the world. Despite the abundance of digital solutions for preventing future pandemics, the world remains unprepared. A specific focus in this study is the speed of response enabled by the social aspect of human-computer interactions (e.g. social machines) and the increased connectivity of humans and devices (e.g. IoT). ## The role of connected devices and social networks The Internet of Things (IoT) refers to technologies with sensing, networking, computing, information processing and intelligent control capabilities. IoT is commonly used to describe various smart objects and things, with computational abilities, that exchange information, potentially even on a global level, perceiving the world as one adaptive and interconnected system [bib_ref] Where the smart things are: social machines and the internet of things, Smart [/bib_ref]. This communication and exchange of information is done via different protocols including WAN, LAN/LoRaWAN, Bluetooth, Z-Wave, INSTEON, ZigBee, Wi-Fi, 4G and 5G. When social networks are integrated with the IoT, a new concept emerges, named Social Internet of Things (SIoT). The SIoT potential architecture, policy and network structure have already been tested with simulations [bib_ref] The social internet of things (SIoT) -when social networks meet the internet..., Atzori [/bib_ref]. These simulations show that SIoT depends on the type of relationship (e.g.) Ownership object relationship (OOR); Social object relationship (SOR); Co-work object relationship (C-WOR); Co-location object relationship (C-LOR); and Dynamic Social Structure of Things (DSSoT). The 'Social Collaborative Internet of Things' (SCIoT) goes even further in respect that social objects are grouped and collaborate by interacting and sharing [bib_ref] When social objects collaborate: concepts, processing elements, attacks and challenges, Khan [/bib_ref] , in other words, working as cognitive coupled systems. These concepts emerge as values in the advancements of smart healthcare monitoring and management systems that are using big data analytics, such as iHealth and m-Health [bib_ref] Erratum to 'smart health monitoring and management system: toward autonomous wearable sensing..., Sadia [/bib_ref]. The diversity of SIoT implementations points to problems of scale and interoperability in terms of aggregation of data for responding to a pandemic. # Methodology This is a review paper, investigating different approaches taken across the world in managing the COVID-19 pandemic with digital technologies. To forecast events, digital technologies currently use artificial intelligence (AI) to process large amounts of data. However, AI applications require narrowly defined use cases and large training datasets to 'teach' the app how to forecast. This would result in a slow adaptability of AI to managing a new and fast-changing pandemic. To use AI in pandemic management, social machines and connected devices will require a different approach to AI training, for example, selective re-purposing of AI systems already trained for other uses, like temperature measurement. A social machine is defined as 'an environment comprising humans and technology interacting and producing outputs or action which would not be possible without both parties present'.As we have seen with most mobile app concepts for pandemic management, they depend on human and technology interaction at scale, that is, with mass participation by large segments of the population. To achieve the targets of the UK's digital contact tracing programme, for instance, it was anticipated that a minimum adoption rate of 60% 2 of the UK population was needed. [bib_ref] The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest..., Peeri [/bib_ref] However, where such apps require the capture and sharing of sensitive data with various entities such as the government, potential users can become wary and reluctant to participate. One study found that over 70% citizens were unwilling to download contact tracing apps. [bib_ref] Response to COVID-19 in Taiwan: big data analytics, new technology, and proactive..., Wang [/bib_ref] Since this data is already shared by many humans on social media and mobile apps, this opens up questions on how to design a privacy preserving contact tracing app that ensures that a sufficient percentage of humans will download the pandemic management app. To investigate this, we refer to social science methods and relate the concept of social machines, with digital health and pandemic management. An additional approach is to use standard open-source intelligence (OSINT) to gather data that is available in the public domain. [bib_ref] Where the smart things are: social machines and the internet of things, Smart [/bib_ref] This includes searches to public repositories of Internet-exposed and vulnerable IoT devices (for instance, shodan.io). ## Social machines in digital health for pandemic management Global pandemic management requires fast data and knowledge acquisition with real-time visualization of results. This can be achieved by interactive spatial transmission and social management of data in geographic information systems [bib_ref] Chinese experts' consensus on the internet of things-aided diagnosis and treatment of..., Bai [/bib_ref]. This relates to the 'Social Internet of Things' (SIoT), which integrates people and smart devices interacting within a social structure of IoT, often through IoT platforms [bib_ref] A gap analysis of internet-of-things platforms, Mineraud [/bib_ref] [bib_ref] Business models for the internet of things environment, Glova [/bib_ref]. The 'Social Collaborative Internet of Things' (SCIoT) represents a more advanced form of SIoT where social objects collaborate, interact and share information, creating cyber-physical ecosystems [bib_ref] Cognitive friendship and goal management for the social IoT, Kasnesis [/bib_ref]. Social machines are human-machine communities on the web (e.g. Wikipedia). These often change, like living hybrid organisms, and may be represented as cyber-physical and socio-technical systems [bib_ref] Observing social machines part 2: how to observe?, De Roure [/bib_ref]. Such systems can be observed through a plurality of archetypal narratives [bib_ref] Archetypal narratives in social machines: approaching sociality through prosopography, Tarte [/bib_ref]. The SIoT utilizes users' needs, preferences, social drives and the surrounding environment to generate situation-aware services [bib_ref] Towards a dynamic discovery of smart services in the social internet of..., Hussein [/bib_ref]. This refers to cognitive reasoning within temporary social structures, combining users, objects and services in the form of a 'Dynamic Social Structure of Things' [bib_ref] Towards a dynamic discovery of smart services in the social internet of..., Hussein [/bib_ref]. Cognitive collaboration between connected devices can be achieved by combining machine learning with middleware technologies [bib_ref] Cognitive friendship and goal management for the social IoT, Kasnesis [/bib_ref]. Some studies even suggest the creation of emotional models for human-machine interactions [bib_ref] Human machine interactive system on smart home of IoT, Kun-Kun [/bib_ref] [bib_ref] The cluster between internet of things and social networks: review and research..., Ortiz [/bib_ref] , as expressed in [fig_ref] Table 1: Evolution of the Social Collaborative Internet of ThingsThe evoluƟon of Social CollaboraƟve... [/fig_ref]. Such human-machine interactions could provide valuable feedback for pandemic monitoring apps. The social networks described in [fig_ref] Table 1: Evolution of the Social Collaborative Internet of ThingsThe evoluƟon of Social CollaboraƟve... [/fig_ref] have large numbers of participants and can provide answers to complex problems [bib_ref] The social internet of things (SIoT) -when social networks meet the internet..., Atzori [/bib_ref]. Hence, when designing pandemic monitoring apps, we should focus on the value of this human-computer interaction for large numbers of participants and a wide range of devices. An approach to managing new mass surveillance technology that links personal data would be to enable the consolidation of connected devices into virtual objects for remote access and control. This could create a dynamic virtual network connecting different domains and facilitating sharing of resources in a cloud environment [bib_ref] Cloud based IoT network virtualization for supporting dynamic connectivity among connected devices, Ullah [/bib_ref]. This would allow us to initiate the development of a truly global response and monitoring of pandemics. ## Social collaborative internet of things and artificial intelligence The SIoT opens new opportunities, such as fast and diverse application development though a dynamic end-to-end connection between devices. For example, there is already a consensus among Chinese experts that Internet of Things (IoT) can help in diagnosis and treatment of the pandemic [bib_ref] Chinese experts' consensus on the internet of things-aided diagnosis and treatment of..., Bai [/bib_ref]. The SIoT enhances the IoT with improved intelligence, contextawareness and cognitive reasoning [bib_ref] Towards a dynamic discovery of smart services in the social internet of..., Hussein [/bib_ref] , such as an IoT cognitive hierarchy thinking mechanism [bib_ref] Cognitive hierarchy thinking based behavioral game model for IoT power control algorithm, Kim [/bib_ref]. Some include using social network theory for social relations of mobile nodes in IoT [bib_ref] Research on social relations cognitive model of mobile nodes in internet of..., An [/bib_ref]. If we consider the concept of mechatronics, where functionality is transferred from the machine system into an information systems [bib_ref] The internet of things -the future or the end of mechatronics, Bradley [/bib_ref] , then the SIoT already represents a form of cognitive engine. Cognition in the form of an aware-SIoT that perceives time, space and activity, depends on building social relationships between 'things' [bib_ref] Research on social relations cognitive model of mobile nodes in internet of..., An [/bib_ref]. Cognition in SIoT could resolve many social issues. For example, one such issue is incorrect self-diagnosis through personal digital health data. This creates increased and sometimes unsustainable demand for medical practitioners. At best such information could be used by clinicians for understanding a patient's lifestyle [bib_ref] The crowd keeps me in shape': social psychology and the present and..., Kleek [/bib_ref]. Cognition in health social machines could prevent the damage of self-diagnosis and instead provide tailored interventions by de-coupling activity data into a consolidated longitudinal archive for an integrated approach to 'intervention, management, mitigation and sense-making' [bib_ref] The crowd keeps me in shape': social psychology and the present and..., Kleek [/bib_ref] , supported with FAIR stewardship for distributed data analytics [bib_ref] FAIR science for social machines: let's share metadata Knowlets in the internet..., Mons [/bib_ref]. With the advancements in machine moral mind [bib_ref] A perceived moral agency scale: development and validation of a metric for..., Banks [/bib_ref] , and criteria to evaluate the health of a social machine [bib_ref] Working out the plot: the role of stories in social machines, Tarte [/bib_ref] , the interactions with social machines could evolve into a meaningful communication based on moral agency, where humans act as a 'cognitive anchor', noting that even humans need models for mental behaviour [bib_ref] A perceived moral agency scale: development and validation of a metric for..., Banks [/bib_ref]. ## Social collaborative internet of things and quality of experience in digital healthcare The SIoT operates in a way that is similar to how humans interact with social networks. The physical social connections between humans and things represent the baseline for the logical configurations of social communities, involving humans and things [bib_ref] The cluster between internet of things and social networks: review and research..., Ortiz [/bib_ref]. The logical configuration adopts individual social network behaviours into a universal social network of all entities [bib_ref] The cluster between internet of things and social networks: review and research..., Ortiz [/bib_ref]. The IoT contains many aspects of social networking, where value emerges from the exchanges between users and devices, enabling service that would be of more interest because of interactive filtering (e.g. embedded systems gathering and analysing data before communicating to social networks of people and devices), to provide better quality of experience [bib_ref] The cluster between internet of things and social networks: review and research..., Ortiz [/bib_ref]. Whilst it is likely that during rapidly unfolding crises, aspects other than service design will be given priority in the design and deployment of new digital interventions, it is well established that ease of use and user experience, combined with a perceived value for participants, could increase uptake and adherence, resulting effectiveness of such interventions. Moreover, since the effectiveness of such interventions depends on high rates of user adoption, rapid uptake and participation are crucial. Thus, systems designed for gathering personal data during pandemics should provide value and quality of service to encourage more participants. Participants could also be encouraged by different systems of trust. There are various schemes of trust, with diverse trust scaling, dimensions, inferences and semantic meanings. For example, for trust in social networks, Amazon and eBay use star ratings; P2P networks measure quality of downloaded files and speed. Some medical devices are considered IoT devices. There are many challenges identifying what closed or open source software is running in medical devices and other IoT devices in the industry. The list of software used in any device (including non-IoT devices) is referred to a software bill of materials (SBOM). Vendors and consumers lack awareness of not only the software components they are using and adopting but also the underlying security vulnerabilities that affect such software. The National Telecommunications and Information Administration (part of the United States Department of Sensing the local environment using local informaƟon Sociality -connecƟvity Commerce) is leading an effort to provide guidelines to producers and consumers of technology on how to create effective SBOMs that can help identify the presence of known security vulnerabilities. [bib_ref] The social internet of things (SIoT) -when social networks meet the internet..., Atzori [/bib_ref] This effort is divided into four major working groups: framing; awareness and adoption; formats and tooling; and healthcare proof of concept. The healthcare proof-of-concept working group created a report that provides information on SBOM creation by the MDMs; consumption by the healthcare delivery organizations (HDO); and the exercising of the procurement, asset management, risk management and vulnerability management use cases. Challenges and areas for improvement were identified and documented around an absence of naming standards, partial version information, vulnerability vs exploitation qualification, configuration vulnerabilities, no authoritative end of life database and lack of patching level data. [bib_ref] When social objects collaborate: concepts, processing elements, attacks and challenges, Khan [/bib_ref] Pandemics and socio-economic hardships COVID-19 has triggered a significant shock in the economy with social distancing and quarantine policies triggering economic recession. Economic hardship could harden the impact of pandemics, and digital technologies provide some solutions for increasing economic value during global pandemics. While current focus is on pandemic management, with the increased focus on digital management of the COVID-19, significant focus is also placed on connected devices and IoT. Prior to the pandemic, it was estimated that IoT will create a value in 2020 projected at 1.9 trillion USD [bib_ref] Business models for the internet of things, Dijkman [/bib_ref] with connected IoT devices reaching 20.8 billion in 2020 [bib_ref] Prototyping business models for IoT service, Ju [/bib_ref]. These connected devices and social networks enable data collection and exchange that was not possible during previous pandemics, and this represents a different healthcare business ecosystem. Healthcare business models need to adapt accordingly to secure ethical access to this new value. Some of the areas of focus to secure ethical access to this new value are in monitoring and control, big data for business analytics, information sharing and collaboration [bib_ref] The internet of things (IoT): applications, investments, and challenges for enterprises, Lee [/bib_ref] , among many other value areas. Harnessing the IoT potential also requires a digital strategy; for example, the cloud offers value in high-scale real-time analytics of big data, and IoT middleware offers lightweight real-time analytics [bib_ref] State-of-the-art, challenges, and open issues in the integration of Internet of things..., Díaz [/bib_ref]. Cloud IoT also enables a vision for worldwide IoT integration [bib_ref] Internet of things (IoT): a vision, architectural elements, and future directions, Gubbi [/bib_ref]. Apart from the medical and business side, there is also a social value from IoT social connection and networking, which can be explored by combining social computing with opportunistic IoT, for analysing the physical and digital social communities simultaneously [bib_ref] Opportunistic IoT: exploring the harmonious interaction between human and the internet of..., Guo [/bib_ref]. By adding intelligence in objects, smart objects collect data, interact and control the physical world while symbiotically sharing data and being interconnected with other things [bib_ref] The internet of things vision: key features, applications and open issues, Borgia [/bib_ref] , though a diverse set of communication technologies that enable remote management [bib_ref] The internet of things vision: key features, applications and open issues, Borgia [/bib_ref]. ## Cyber risk from digital solutions in pandemic management With the integration of large numbers of social IoTs in a digital healthcare design, a large data distribution platform can be subjected to a relatively simple large-scale attack, triggering a huge economic cost and even loss of life [bib_ref] Distributed attack detection scheme using deep learning approach for internet of things, Diro [/bib_ref]. These emerging risks from IoT in healthcare ecosystems need to be understood in the digital solutions design process. One way to understand these risks is through existing reference architectures for attack surface analysis. Cloud IoT ecosystems are seen as pervasive and disruptive, enabling many different applications that are gaining momentum, but with many issues surrounding privacy and security [bib_ref] Integration of cloud computing and internet of things: a survey, Botta [/bib_ref]. There are however existing cyber risk assessment approaches for smart systems [bib_ref] Smart cyber-physical systems: beyond usable security to security ergonomics by design, Craggs [/bib_ref] , for IoT systems [bib_ref] Security risk assessment in internet of things systems, Nurse [/bib_ref] and for cloud provider environments [bib_ref] Cyber risk assessment in cloud provider environments: Current models and future needs, Akinrolabu [/bib_ref]. The use of these could enhance the privacy of digital heath solutions. SIoT is in the same risk category. SIoT objects can anonymously establish communications and cooperate opportunistically with neighbouring IoT devices, but trust management in SIoT is a concern [bib_ref] When social objects collaborate: concepts, processing elements, attacks and challenges, Khan [/bib_ref]. Therefore, existing performance metrics for security operations analysis [bib_ref] Challenges and performance metrics for security operations center analysts: a systematic review, Agyepong [/bib_ref] and failsafe recommendations [bib_ref] Failures from the environment, a report on the first FAILSAFE workshop, Breza [/bib_ref] should be consulted in the design of digital heath solutions. ## Case study of digital solutions in pandemic management Two data sources were used extensively in case studies: (1) the Google Scholar search engine and (2) a critical appraisal of Publons' new COVID-19 publication index that investigates sound scientific practices of preprints and papers. As COVID-19 is a global and fast-spreading pandemic, as visualized in , the case study research is not focused on investigating individual region responses in great detail. We designed the diagram in , with data from this study, using the 'draw.io' open access web software. [bib_ref] Erratum to 'smart health monitoring and management system: toward autonomous wearable sensing..., Sadia [/bib_ref] The different lines represent the spread of COVID-19 regionally, nationally and internationally, leading to a global pandemic. The COVID-19 pandemic has spread globally (demonstrated conceptually in , without reliance on concrete data) even before most governments realized the need to act; for example, at the time of writing, the first known case in France was in December 2019, almost a month before the French government announced the first case in France. [bib_ref] Chinese experts' consensus on the internet of things-aided diagnosis and treatment of..., Bai [/bib_ref] Therefore, studying regional approaches in detail at this stage, when we do not truly understand the data, would probably not produce the results that we require. Instead, focus was placed on the regions that performed best during the initial outbreak, with the objective to gather and accumulate knowledge on the effectiveness of different methods and with the aim of making recommendations for replicating this success in the second wave. ## The case of covid-19 in taiwan -role of social machines in digital democracy The Taiwanese culture of civic technology, a fusion of technology, activism and civic participation, created a strong response to COVID-19. The vTaiwan digital democracy platform [43] presents a decentralized community of participants. The platform hosts social machines that uncover a problem and then help participants to understand this problem, even while the situation is changing daily or hourly. Taiwan implemented actions (interventions) with great speed through the platform . Some of the actions were aimed at reducing panic buying, such as the application programming interface (API) that provided real-time, location-specific data to the public on critical items' availability. To design the interactive face mask maps, for example, the digital minister worked with hacktivists through a digital chatroom. A second example of success was a bottom-up information sharing platform on which individuals created a strong participatory collective action for sharing real-time reports about symptoms using social smartphone apps, call-in lines and other approaches. The results were integrated with location history by community-created apps, enabling individuals to track and compare their exposure. This approach supported proactive behaviour, where people who were concerned about being exposed had a tool to determine if they should selfisolate prior to developing symptoms. The authorities also had a tool to monitor people in self-isolation with a mobile phone-based 'electronic fence' that traces mobile phone signals to track location and follow up with two phone calls every day to make sure people stay home. The Taiwanese system sends alerts to police if self-quarantined people leave their home, and the police would contact or visit the person within 15 min of the alarm being triggered. The Taiwanese 'electronic fence' system could be the first in the world designed for this purpose. [bib_ref] A gap analysis of internet-of-things platforms, Mineraud [/bib_ref] The success factor for such large-scale social coordination was the common-sense approach and the privacy protection of participating individuals. This platform reduced economic impact greatly, through successful containment that avoided social distancing measures. People either self-isolated, avoided or disinfected contaminated locations, depending on their location history review. The community data enabled more targeted responses that have proven more effective than in all other economies, including the USA, the EU and China, at least in the first wave. The case of Taiwan has proven that social inputs and coordination can prove more effective in resolving complex fast-evolving problems than centrally run top-down approaches that take a while to deliver and, in some regions, lack buy in by the population. Further, the community-driven development of software tools in this case was proven to be fast and precise, driven by society participation. This review paper is focused on the human-computer interactions in pandemic management, where technology creates value when built into social machines, where a large number of participants adding input in mobile apps. However, it is worth mentioning that AI and ML can also add value in purely digital approaches to solving problems. AI/ML applications generally work best with narrowly defined use cases accompanied by large, hand-built training datasets to 'teach' an application how to execute against its use case. This results in slow adaptability of AI/ML to new opportunities, unless an existing capability can be re-purposed. For example, consider the Kogniz Health product 11 -a stand-alone infrared digital camera with an integrated ML trained to identify passers-by who show signs of fever and send alerts in real time via a variety of standard messaging tools. The ML capability is possibly re-purposed from an existing ML use case to monitor and alert on temperature variations beyond acceptable limits in an industrial setting. This approach could work in advanced economies because of partially existing infrastructure, strong know how and technological/financial strength. But it is difficult to see how these tools can be made operational in developing countries, at the required speed. ## The case of covid-19 in the uk Given the lack of treatment such as a vaccine for COVID-19, the world's current focus is on global surveillance, and IoT and other new technologies including thermal cameras or IoT sensors [bib_ref] On the coronavirus (COVID-19) outbreak and the smart city network: universal data..., Allam [/bib_ref] , and face recognition [bib_ref] Digital technology and COVID-19, Ting [/bib_ref] , that can play a crucial part in mapping the spread of infection, and in detecting disease early as part of smart systems with near real-time data tracking and alerts [bib_ref] The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest..., Peeri [/bib_ref]. Currently IoT-enabled drones are used for public surveillance, enforcing quarantines and even for faster disinfecting with agricultural drones. IoT can be used for enforcing compliance of infected people in quarantine, by monitoring and tracing potential infections caused by breach in quarantine. The speed and scale of IoT connectivity could enhance the UK's National Health Service (NHS) ability to monitor high-risk patients in home quarantine. This is currently done with healthcare workers going door-to-door, posing a risk to them, and using up resources for home checks that could turn out to be unnecessary. IoT real-time live cameras could be used for patient checks, cloud databases could be used for patients to upload their temperature measured on home thermometer, and drones could be used to check temperature with infrared cameras. The aggregated IoT mobile data from infected patients can be used for tracing people who might have been in contact with infected patients. The IoT is already used for remote monitoring of in-house patients with serious conditions. Self-reporting apps such as the 'C-19 COVID symptom tracker' enable quick buildup of maps and other data that support people to prevent infection and medical professionals to plan their response. One of the main benefits from app design that operates as a social machine is the speed of deployment and data collection. For example, the UK 'Covid Symptom Tracker' app reached 700,000 downloads and app registrations in 24 h [bib_ref] Covid-19: researchers launch app to track spread of symptoms in the UK, Mayor [/bib_ref]. It is almost impossible to gather such large datasets with any of the traditional data collection tools. But societal participation in data collection, during times of crises, seems to be producing relevant data at a very fast rate. Earlier attempts were different in approach. For example, a mobile phone application called FluPhone Appused Bluetooth and Wi-Fi signals to track human interactions and asked users to report symptoms. The data collected was used for understanding how diseases spread in complex human network structures, using information such as how frequently people meet and how much time they spend together. One of the main weaknesses in this approach is that smartphones can accurately determine location between 7 and 13 m [bib_ref] Smartphone GPS accuracy study in an urban environment, Merry [/bib_ref] , and COVID-19 spreads in people that are within less that 2-m proximity. Tracing Bluetooth and Wi-Fi location also does not always help with mapping objects in movement (e.g. flights, trains and buses). ## The case of covid-19 in china -role of iot in social machines The Chinese approach for compulsory registration of phone numbers to an app, prior to using public transport, and allocating colour codes through social media apps, could be more effective in using big data for automated forecasting of contagion risk, but it triggers privacy concerns. The Chinese approach is enabled through the wallet app Alipay and the social media, messaging and mobile payment app WeChat. The apps display a code that can be read by smartphones: green means unrestricted and yellow or red implies a need to selfquarantine or be in a supervised quarantine. Codes are based on tracing location history to determine contacts with outpatients or people hospitalized in the past 14 days and are refreshed every day at midnight. Many restaurants, coffee shops and shopping malls require to see a green code to enter. But there are already concerns about glitches and incorrect codes that ban people from travelling or entering public spaces, While the Chinese approach for digital epidemic control has triggered privacy concerns and mistrust, similar public response can be expected in the EU and USA where people have even lower level of trust in their governments. This mistrust can be fuelled even more with the increased attacks against individuals working from home during the pandemic. [bib_ref] Cognitive friendship and goal management for the social IoT, Kasnesis [/bib_ref] There is also a big difference in how such cyber-attacks are mitigated. One malware sample we discovered was a wiper designed to destroy infected systems. The malware was aimed at people working from home but also at healthcare organizations. The filename '冠状病毒.exe' which translates to 'coronavirus' uses several techniques to delete data from both the file system and registry in an attempt to disrupt system operations. The Taiwanese approach has on the other hand caused far less mistrust despite involving big data analytics, the integration of national health insurance databases, access to travel history from individual's location databases, code scanning and online reporting. The main difference between the EU, the USA, China and Taiwan on the other hand is the difference in public communication strategies. The case of COVID-19 in South Korea -Social machines and IoT in smart cities South Korea was one of the countries where COVID-19 started early in the global outbreak, and their experience with SARS has proven effective in lowering the death rate through strict quarantine and fast testing. South Korea deployed a tracking app called Corona100m (Co100), informing people of known cases within 100 m of their location. [bib_ref] Observing social machines part 2: how to observe?, De Roure [/bib_ref] Further tracking measures included a 'smart city technology system' that uses surveillance camera footage and credit cards transactions to track past movement of infected patients. 14 These tools are in addition to the Coronamap 15 website tracing the location history of the infected and the information search engine Coronaita 16 designed specifically for finding information on infected areas. ## The role of tech companies in covid-19 pandemic management The Chinese response to COVID-19 has shown that information systems for pandemic management, in terms of database and spatial analysis and mapping, can be designed and made operational fast but is limited by the commercial software that is in existence. Some of the main challenges are the largescale people flow globally, the long incubation period of this virus, lack of symptoms in some of the infected, virus reoccurrence after recovery, evolving nature of the virus targeting different DNAs and the different level of impact on individuals. If pandemic tracking information system is designed to operate regionally, its functions are limited in global pandemic prevention monitoring. ## Statistical bibliometric analysis of scientific research records Two different software tools were heavily used for a bibliographic review of the types of data used in pandemic management: (1) R Studio was used with the 'bibliometrix' [bib_ref] Bibliometrix: an R-tool for comprehensive science mapping analysis, Aria [/bib_ref] package to analyse the large sets of data records, and (2) a computer programme for bibliometric mapping called VOSviewer [bib_ref] Software survey: VOSviewer, a computer program for bibliometric mapping, Van Eck [/bib_ref] was used for graphically representing large data records in a bibliometric map. The Web of Science Core Collection was identified as the data source for the two different software tools. The data search parameters were narrowed to search for articles on COVID-19 in 2020. Then, a historic search parameter was used to identify methods applied in research on previous pandemics. From the first search result, we searched for 'COVID-19', and out of 2264 records, we used the Web of Science filter and downloaded the most relevant 500 records. When analysed with the R 'bibliometrix' package, we selected two most striking figures. In , we can see a three-fields plot of keywords, topics and countries. To compare the three-fields plot analysis with alternative mapping of scientific research on COVID-19, we designed a graph by corresponding author countries . To compare these results, with the entire 2254 records on the Web of Science Core Collection, we used the Web of Science result analysis tool and found similar results as seen in [fig_ref] Figure 4: Web of Science-results analysis tool management [/fig_ref]. Although in Figs. 2 and 3, China is shown as the dominating country in the scientific research on COVID-19, we compared the three-fields plot analysis, by using the same data file of records and designed a collaboration network as a social structure [fig_ref] Figure 5: Collaboration network as a social structure of COVID-19 scientific research records in... [/fig_ref]. What is clear from [fig_ref] Figure 5: Collaboration network as a social structure of COVID-19 scientific research records in... [/fig_ref] is that the UK is performing strongest in collaborative research on COVID-19, and this collaboration is strongly represented between the UK, the USA and Canada. We continued this analysis by doing a historical search on the Web of Science Core Collection for scientific research on global pandemics. This resulted with 3708 records, and we used the Web of Science tool for research analysis (see [fig_ref] Figure 6: Historic analysis of scientific research on global pandemics 1900-2020 [/fig_ref]. The historic analysis showed that countries that have led the scientific research on global pandemics in the past are not represented as strongly as China in the scientific research on COVID-19. Since China was first affected in this global pandemic, we could speculate that regional research effort is driven by the effect of the pandemic. It is possible that other countries would pick up and start producing more scientific research, but such comparison can only be confirmed when data becomes available. At present, these graphs represent statistical analysis of the current data, as a snapshot in time, compared with historical analysis in [fig_ref] Figure 6: Historic analysis of scientific research on global pandemics 1900-2020 [/fig_ref]. What is concerning from the results of this statistical analysis of scientific research is the extremely low record numbers ## Types of digital data in pandemic management In this section we detail our search for scientific research using digital data. Firstly, such data does exist. Digital healthcare (e.g. e-Health, m-Health, Telemedicine) includes management of large, heterogeneous, distributed, multiscale and multimodal datasets distributed across edge-fog-cloud healthcare paradigms. Complementing such paradigms, smart cities generate vast amounts of data, captured and stored in different heterogenous formats that need to be transformed to be of analytical value [bib_ref] Data integration for smart cities: opportunities and challenges, Raghavan [/bib_ref]. IoT and big data analytics are emerging trends in new biomedical and healthcare technologies [bib_ref] Emerging trends in IoT and big data analytics for biomedical and health..., Banerjee [/bib_ref]. These various different types of data need to be integrated to ensure fast and effective response during pandemic Although there are concerns about data privacy, there are many types of data that are already shared openly in different community settings (e.g. skill set, goods sold, vehicles movement). Some personal data needs to be evaluated with specific privacy requirements in consideration, but there is also a lot of data shared in open community marketplaces [bib_ref] A new paradigm for a marketplace of services: smart communities in the..., Eltoweissy [/bib_ref]. Focusing on one specific data type (e.g. big data) would not necessarily result in low latency. Adding to this, the peerreviewed scientific research on individual data sources is still in its infancy, and the COVID-19 pandemic is already causing deaths globally. Currently, there are only five scientific publications on the Web of Science Core Collection under the search on 'COVID-19 and big data'. We reviewed all five publications, and only one actually discussed big data solutions. This one publication is only a viewpoint, not a research article. A similar search on 'pandemics and big data', using historical records since 1900 (to 2020), produced only 59 records. On the other hand, search results on 'big data' alone produced 88,410 records. It is unclear if this result is caused by lack of funding or lack of interest in the past on this research topic. Hence, we continued with different search parameters, and here we discuss few different types of data that need to be integrated in the pandemic management analytical processes, but the scientific research on these topics is very limited. Time-stamped data refers to data that includes the event time (the time of the event) and processed time (the time data was processed). By recording the time of capture and time of collection, data analytics could recreate location journey, by replaying people's steps and even derive findings on estimating or predicting best future actions. Our search on the Web of Science Core Collection, on the topic of 'COVID-19 and time-stamped data', resulted in no results, while the search of historic records, from 1900 to 2020, on 'pandemic and time-stamped data', resulted in only 3 records. Genomics data that involves analysing the DNA of patients could identify new drugs and improve healthcare with personalized treatment. But converting genomic data into value is challenging, because of the large volume of data that requires significant processing resources, combined with the need to unify diverse teams from bioinformatics data specialists, to clinical specialists on the front line. Nevertheless, significant progress has already been made. Sequencing the first genome took longer than a decade. With current tools, it takes just 2 days despite these limitations in handling large data. Despite these advancements, our search on the Web of Science Core Collection, on the topic of 'COVID-19 and genomics data', resulted in 0 results, while the search of historic records, from 1900 to 2020, on 'pandemic and genomics data', resulted with 43 records. A similar search on 'COVID-19 and high-dimensional data' also resulted in no records, and the search of historic records, from 1900 to 2020, on 'pandemic and high-dimensional data' resulted in only two records. However, to quote a famous aphorism frequently used in this context [bib_ref] Statistics notes: absence of evidence is not evidence of absence, Altman [/bib_ref] , the 'absence of evidence is not the same as evidence of absence'. We are aware that the data is currently incomplete so we cannot assume for sure that the research is not happening. We will run this analysis again in a year and see how it has changed and how it compares to previous pandemics. Healthcare data contains a large number of interrelated variables (e.g. weight, blood pressure, cholesterol levels). To investigate the connection between intraday physical activity and sleep, actigraphy data was applied to a Bayesian framework for function-on-scalars regression [bib_ref] Bayesian function-on-scalars regression for high-dimensional data, Kowal [/bib_ref] , resulting in many proposed predictors. Similarly, it is possible to check an individual for the expression of thousands of genes, resulting in millions of possible gene combinations, some of which may be predictors. This is described as highdimensional data, where the number of features exceeds the number of observations. To observe high-dimensional data for pandemic management, one would need to analyse multiple measured and recorded parameters including, for example, immune system status, genetic background, diagnosed diseases, operations, treatments, blood analysis, nutrition and alcohol-tobacco drug consumption. Open sharing of such personal medical data would require people to trust in technologies such as the secure multiparty computation (also known as privacypreserving computation) [bib_ref] The cluster between internet of things and social networks: review and research..., Ortiz [/bib_ref] and differential privacy. [bib_ref] Cloud based IoT network virtualization for supporting dynamic connectivity among connected devices, Ullah [/bib_ref] Such technologies could enable a broader interpretation, based on trust in privacy preserving technologies. More work needs to be done on identifying and promoting the benefits for private individuals to start sharing openly medical data. Genetic data and personal biodata privacy are evaluated with a unique moral obligation for protecting patients' dignity, and there are ethical limits on how such data can be shared [bib_ref] The ethical limits of blockchain-enabled markets for private IoT data, Ishmaev [/bib_ref]. Analytic transactional (transact and analyse) data comes in handy in resolving this issue in the healthcare environment. Analytic transactional data enables shifting real-time processing with new metrics and in-memory computing. Instead of using one technology to run applications, and separate technology to run batch analysis, creating linked layers and a patched system, analytic transactional data analysis uses a single layer for transactions and analytics. This approach would enable monitoring health indicators in real time while performing other functions simultaneously. Despite its scientific usefulness, the search on 'COVID-19 and analytic transactional data' and the search of historic records, from 1900 to 2020, on 'pandemic and analytic transactional data' both resulted in no records. The search was repeated with multiple terms (e.g. 'analytic transactional', and 'translytic' among other terms). Spatiotemporal data can be analysed for building an interactive view of data points in similar and different regions, to analyse data from localized areas or to map and compare spatial distribution in individual regions [bib_ref] GeoBrick: exploration of spatiotemporal data, Park [/bib_ref]. In previous pandemics, spatiotemporal analysis has been used to identify cholera hotspots in Zambia [bib_ref] Identification of cholera hotspots in Zambia: a spatiotemporal analysis of cholera data..., Mwaba [/bib_ref]. With COVID-19 spreading so fast, the scientific peer-review process is struggling to catch up. Currently, there is only one scientific publication on the Web of Science Core Collection under the search on 'COVID-19 and spatiotemporal data'. On the other hand, there are only 24 records on the Web of Science Core Collection under the search on 'pandemic and spatiotemporal data', which means that scientific research is lacking on this topic. Since our search of established peer-reviewed scientific research on the Web of Science Core Collection resulted with too few records to conduct statistical analysis, in the next section, we instead applied a quantitative case study research methodology. In the case study research, we used other search engines, such as Google Scholar. However, the consistently low records on the Web of Science Core Collection present an overwhelmingly strong argument that highly ranked scientific research, in a sufficient quantity for statistical review and analysis, is currently missing. ## Summary of digital solutions for covid-19 and future pandemic management The current digital health infrastructure requires enhancements to support medical efforts in COVID-19 and future pandemics management. Such enhancements are not only required in the field of artificial intelligence algorithms but also in digital infrastructure for data collection in relation to current and past location monitoring, disease transmission forecasting and health monitoring. This is similar to enhancements of infrastructure as response to other natural disasters (e.g. floods, earthquakes). Fortunately, digital technologies have advanced significantly since the last pandemic, presenting a variety of digital solutions that could be adopted for pandemic management relatively easy. The COVID-19 crisis also changed the perception of medical data sharing, leading to wide acceptance of new mobile apps designed for various pandemic management functions, all based on some form of sharing personal data. Just a few months ago, it would have been unprecedented to even think about one million downloads for a mobile app that requires sharing your personal medical data, including current and past location history. There seems to be a consensus among governments about the benefits of such mobile apps. There are, however, some measures that are operational in individual regions, but have not even been considered in others. In [fig_ref] Figure 7: Digital solutions for pandemic management [/fig_ref] , these tools and measures are integrated, to design a more comprehensive data collection and migration, for epidemic monitoring of regional risk trends and indices of highrisk people or areas. We designed this diagram with data from this study, using the 'draw.io' open access web software. [bib_ref] Cognitive hierarchy thinking based behavioral game model for IoT power control algorithm, Kim [/bib_ref] In the past decade, there was a fast innovation of new digital technologies. This resulted in the new concept of digital health, on which some of the solutions in [fig_ref] Figure 7: Digital solutions for pandemic management [/fig_ref] are based. Although these digital solutions are already operational and can easily be adopted for pandemic management, without proper planning significant challenges could emerge. Some challenges could relate to capturing duplicate data, hence adding significant workload on medical workers that collect, manage and use the data. The design of these digital health solutions separately could lead to lack of interoperability when their integration is crucial for making evidence-based decisions. Individual design could also lead to complexity, for example, hindering the ability of medical professionals to use digital health systems. Open standards for data structure and interoperability in this discipline would help. While pandemics require a global response, not all countries have the capacity to govern the epidemic response in line with privacy preserving policies. Technologically advanced nations have the advantage in this field and could be faster to use this leverage to advance the global digital health management. Technologically advanced nations could expand these interoperable data systems and tools, strengthening data governance and building capacity to collect and interpret data. However, digitalisation on its own does not improve the health outcome. A coordinated effort is needed that includes both public and private sectors. Past cases have proven that Zika and dengue outbreaks can be predicted within 400 m, months in advance and with 88.7% accuracy, while in Vietnam, an electronic surveillance platform is operative for 44 communicable diseases and syndromes in 63 provinces and 711 districts. [bib_ref] Research on social relations cognitive model of mobile nodes in internet of..., An [/bib_ref] These examples confirm that interoperable digital health systems lead to faster detection and response and increased preparedness. We review in more detail these interoperable digital health systems as shown in [fig_ref] Figure 7: Digital solutions for pandemic management [/fig_ref]. Case study on social networks operating as social machines in digital healthcare Social networks with large numbers of participants can provide answers to complex problems [bib_ref] The social internet of things (SIoT) -when social networks meet the internet..., Atzori [/bib_ref]. The virtualisation of IoT devices into virtual objects for remote access and control creates a dynamic virtual network connecting different domains, facilitating sharing of resources in a cloud environment [bib_ref] Cloud based IoT network virtualization for supporting dynamic connectivity among connected devices, Ullah [/bib_ref]. This creates opportunities for fast and diverse application development through a dynamic end-to-end connection between devices that include those following. ## Social media citizen's advice A social media citizen's advice type system could be designed to connect people with skills and expertise to those who need free access to the advice. This could include legal advice, health, well-being and nutritional advice, technical home computing advice, DIY and small-scale problem-solving engineering advice. This could link to a new app in a format allowing talking about points of concern and should include an option for sharing symptoms and predicted evolution. [bib_ref] Research on social relations cognitive model of mobile nodes in internet of..., An [/bib_ref] https://www.csis.org/analysis/can-digital-health-help-stop-next-epidemic Identify fake data Social media should start classifying and flagging misinformation relating to virus spread; and healthcare systems that monitor these social networks or that directly collect reports from patients should include automated analysis to identify postings concerning symptoms. Fake data in digital systems based on human input is a well-studied subject [bib_ref] Complex coupling in cyber-physical systems and the threats of fake data, De Roure [/bib_ref] , and digital health systems should be based on existing recommendations. Seamless global sharing and processing of geographical data that preserves privacy The complex and diverse set of data sharing restrictions imposed by countries, regions and companies, sometimes embodied as laws, standards or policies, need to evolve into internationally supported and consistently standardized protocols for the seamless sharing of data, especially where data is kept private for geo-political or commercial reasons [bib_ref] On the coronavirus (COVID-19) outbreak and the smart city network: universal data..., Allam [/bib_ref]. Such data sharing would enable the development of a large database that might be enhanced with artificial intelligence tools for processing data for early detection and better virus containment. ## Definitions of trust in data collection and analysis during pandemics In some countries, such as the EU and the USA, people have low level of trust in their governments. Hence, gaining trust in digital systems for data collection and analysis during pandemics is important. Trust is described differently in social networks; for example, Amazon and eBay use star ratings, while P2P networks measure quality of downloaded files and speed. There are various schemes of trust, with diverse trust scaling, dimensions, inferences and semantic meanings. ## Population location activity surveillance A mobile phone app can be used to track down the people who have been in close contact with those diagnosed with COVID-19. An app could use contact information (such as calendar entries/social nets) to track potential infections. In addition, cloud, IoT and social media can be used in combination with a new app, for general population location activity surveillance of quarantined people (by locality) and to design worldwide interactive maps. The cloud enables a vision for worldwide IoT integration [bib_ref] Internet of things (IoT): a vision, architectural elements, and future directions, Gubbi [/bib_ref]. Anonymised IoT medical data sharing though the cloud enables the development of interactive maps of COVID-19 cases worldwide. The cloud and IoT technologies can be used for image or video tracking of (super)spreaders or those regularly flouting social distancing advice. The phone app could enable linking to volunteering groups that allows (verified/tested) people who have got through COVID-19 to be part of an immune volunteer resource pool. In the development of pandemic management interactive maps, the definitions of trust, privacy and security in digital systems for data collection and analysis during pandemics need to be based on globally accepted online standards. ## Local interactive maps A localized COVID-19 mapping system can help residents monitor total cases in their areas, which will enhance value of data sharing and strengthen uptake of government measures. ## Covid-19 pandemic observatory A pandemic observatory can be used for COVID-19 and for fast response in future pandemics. The observatory would be a cloud-based platform designed to ingest, store, share, analyse and visualize COVID-19-related datasets, e.g. to support and integrate data collected through an app that people voluntarily install to allow access and tracking of (a) health and wellbeing details; (b) access to location data; and (c) to insert infection data for mapping behaviour with timeline and geography of the virus (findings might be incubation time; mapping of first symptoms; length of feeling sick; and response to identification of sickness). ## Case study on connected devices operating as social machines in digital healthcare Anonymised real-time data from wearable devices for spatiotemporal mapping and healthcare Wearable devices and other types of healthy sensors measuring heartbeat, blood pressure and body temperature are abundantly present in many urban areas. Future design of such wearables (e.g. Fitbits, Apple Watches) can be adapted to enable body temperature and other symptoms that would enhance early self-diagnosis of viral infections. Allowing anonymised data to be collected, analysed and shared in real time via the cloud can be used for building spatiotemporal mapping and for enhancing the idea of 'connected healthcare', without breaching data privacy and security [bib_ref] On the coronavirus (COVID-19) outbreak and the smart city network: universal data..., Allam [/bib_ref] , by following existing advise on end-user control [bib_ref] X-ray refine, Van Kleek [/bib_ref] and automation control systems [bib_ref] The industrial internet of things (IIoT): an analysis framework, Boyes [/bib_ref]. Future wearables should be encouraged to include skin sensors for temperature monitoring, which would be linked to geo-mapping and local medical or volunteering teams to ensure people are safe and secure. Wearable devices should include automated hand washing assessment on washing hands properly. Wearable sensors can be used to quantify lung volume and functions, or point of care systems could provide care at home and alleviate the pressure on NHS. Respiratory parameters can be estimated using wearable sensors, spirometers, activity sensors and SPO2 sensors to provide early detection and improved prognosis in patients. The goal should be to create value [bib_ref] Value creation for IoT: challenges and opportunities within the design and development..., Lee [/bib_ref] , and it is the design that drives value from connected devices [bib_ref] Design Drivers: a critical enabler to meditate value over the NPD process..., Lee [/bib_ref]. ## Agritech during pandemics Agritech is the use of technology in agriculture, horticulture and aquaculture for improving yield, efficiency and profitability. [bib_ref] The internet of things -the future or the end of mechatronics, Bradley [/bib_ref] During global pandemics, agricultural harvest faces a significant drop in workers. [bib_ref] The crowd keeps me in shape': social psychology and the present and..., Kleek [/bib_ref] Agritech can reduce inputs and increase yields, but adoption is costly, complex and slow. COVID-19 is an opportunity for governments to support big and small agritech companies and new pioneering start-ups [bib_ref] FAIR science for social machines: let's share metadata Knowlets in the internet..., Mons [/bib_ref] to help rapidly accelerate it, not only to help the agriculture industry to survive COVID-19 but also to prepare the industry and develop the technology in anticipations of future global pandemics. # Smart meter analysis Geographical analysis of electricity, water and gas consumption may be used to evaluate adherence to social distancing; with mobile alerts or warnings sent to non-adhering areas. Privacy-preserving measures could include focus on adherence over a large geographical area and not on individual households. Similar analysis is already published on the domestic energy usage patterns during social distancing. [bib_ref] A perceived moral agency scale: development and validation of a metric for..., Banks [/bib_ref] Mobile telemetry for lockdown monitoring Mobile phone networks hold personal mobile phone telemetry that can be used as a real-time data source for feeding AI techniques to identify and send message alerts to those likely to be ignoring the lockdown. Personal mobile phone telemetry can also be used in an app to accompany contact tracing though Bluetooth and Wi-Fi data in identifying those who have come into contact with someone who has COVID-19. In addition, some smart home systems can help with pandemic management. Existing supervised intrusion detection systems [bib_ref] A supervised intrusion detection system for smart home IoT devices, Anthi [/bib_ref] could be adopted to detect home visitors that should be in self-isolation. ## Mhealth and virtual clinics as mobile applications Mobile health (mHealth) [bib_ref] Working out the plot: the role of stories in social machines, Tarte [/bib_ref] is not a new concept. It involves using mobile devices, usually through a secure mobile app, for healthcare management, data sharing and improving patient experience. Apart from mHealth, there are also virtual clinics that can provide personalized medical advice and encourage participatory surveillance for geo-located predictions. Currently operational virtual clinics include PingAn in China.They are effective at reducing costs and saving time and may provide access to medicines through machine dispensers. In developing countries, the cost and time required to build, equip and distribute new machine dispensers may be prohibitive. Adapting the already built and distributed existing food and drink dispensing machines could be faster and more effective. A simple restocking with non-prescription over-thecounter medicines while changing the prices would provide a fast and cost-effective solution. Virtual clinic pharmacy testing kit outcomes should be linked to a central database and any pandemic monitoring system. Virtual clinics should also enable video and supervised tutorials for the self-management (at home) of oxygen therapy, including protocol, sensor use (SPO2) and O2 generation/administration. To be effective in pandemics when most of the population is in self-isolation, virtual clinics should evolve into mobile applications enabling home access. Similarly to the South Korean 'self-quarantine safety protection' app that traces GPS location to track if people are avoiding quarantine and enables self-quarantined people to communicate with medical caseworkers, [bib_ref] Business models for the internet of things, Dijkman [/bib_ref] teleconferencing, or video conferencing, has long been used in telehealth, [bib_ref] Prototyping business models for IoT service, Ju [/bib_ref] also known as telemedicine. It involves a real-time video call, while exchanging other medical data (e.g. blood pressure) required for a medical check. Collaboration apps for virtual meetings are also well established in the medical profession. Collaboration apps enable remote data collection from patients and analysis by medical practitioners based in different geographical locations. Global pandemics have exposed a weakness in the personal protective equipment (PPE) manufacturing and supply chains. Some hospitals have resorted to the use of substandard PPE because they had nothing else to use. Individual GPs have reported using the black market to obtain PPE at a much higher cost. [bib_ref] The internet of things (IoT): applications, investments, and challenges for enterprises, Lee [/bib_ref] Urgent PPE deliveries have also been found of substandard quality and had to be taken out of use. 30 Advice 21 https://en.wikipedia.org/wiki/Agritech 22 https://www.forbes.com/sites/freylindsay/2020/03/24/with-no-eu-workerscoming-the-uk-agriculture-sector-is-in-trouble/#5bd1d684363c 23 https://worldagritechusa.com/agri-tech-start-ups/ 24 https://octopus.energy/blog/domestic-energy-usage-patterns-during-socialdistancing/ 25 https://en.wikipedia.org/wiki/MHealth 26 https://www.mobihealthnews.com/news/asia-pacific/ping-good-doctorlaunches-commercial-operation-one-minute-clinics-china 27 https://www.smartcitiesworld.net/news/news/south-korea-to-step-uponline-coronavirus-tracking-5109 28 https://en.wikipedia.org/wiki/Telehealth 29 https://www.forbes.com/sites/daviddisalvo/2020/03/30/i-spent-a-day-inthe-coronavirus-driven-feeding-frenzy-of-n95-mask-sellers-and-buyers-andthis-is-what-i-learned/#2968466956d4 30 https://www.ft.com/content/f3435779-a706-45c7-a7e2-43efbdd7777b has also been given to medical staff to refuse treating patients if they do not have adequate PPE. [bib_ref] Internet of things (IoT): a vision, architectural elements, and future directions, Gubbi [/bib_ref] [bib_ref] Opportunistic IoT: exploring the harmonious interaction between human and the internet of..., Guo [/bib_ref] Given these widespread concerns about the ability to manufacture and supply sufficient PPE in the face of sudden demand, we should be looking at how robotic technologies could be used to improve the safety of medical professionals during a second wave or in future pandemics. There are three types of healthcare robotics: medical, assistive and rehabilitative. When AI enhanced, robots could attend to multiple patients in quarantined areas, for example, by delivering drugs. Robots could also be enhanced with interactive chatbot, similar to the WHO WhatsApp auto responder, which has the potential to reach 2 billion people. ## Blockchain technology in digital health systems Blockchain technology could enhance the security and privacy of digital health systems while resolving specific problems in pandemic management (e.g. blockchain in virtual pharmacies for panic buying prevention). Dispensing nonprescription medicines (e.g. paracetamol, cough syrup), medical items (e.g. face masks) or household essentials (e.g. disinfectants) through low-tech dispensing machines is a quick and low-cost solution for 24-h available distribution with minimal human interaction. But as we have seen during the initial stages of the COVID-19 pandemic, people behave irrationally during crises and panic buying could lead to just a few people emptying the dispensing machines. In many countries, shops place restrictions on essential items, to preserve stocks from panic buying. Given that virtual pharmacies are a low cost, low human interaction solution, such restrictions need to be implemented through a technology that enables purchase control and monitoring. Panic during the COVID-19 pandemic was only a temporary issue. Therefore, for resolving shortterm issues, purchase control mechanisms should either be low cost or enhance other functions of the systems (e.g. security and privacy). Blockchain technology 33 has been in existence for some time. Open and public blockchain provides security without access control, serving as the transport layer for applications to be added to a network without the separate approval of trust. Design principles have already been proposed for 'tamperresistant gathering, processing, and exchange of IoT sensor data' [bib_ref] Blockchain for the IoT: privacy-preserving protection of sensor data, Chanson [/bib_ref] privacy-preserving blockchain-based sensor data protection systems (SDPS). One of the use cases for evaluating the SDPS is IoT in a pharmaceutical supply chain. We demonstrate in [fig_ref] Figure 8: Blockchain in digital health systems integrated to COVID-19 virtual clinics [/fig_ref] how blockchain could operate in digital health systems. We designed the diagram in [fig_ref] Figure 8: Blockchain in digital health systems integrated to COVID-19 virtual clinics [/fig_ref] with data from this study, using the 'draw.io' open access web software. [bib_ref] Distributed attack detection scheme using deep learning approach for internet of things, Diro [/bib_ref] There are permissionless (e.g. bitcoin) and permissioned blockchains (e.g. Facebook libra). The main difference for digital health systems is that permissioned blockchains are vetted by the network owner, whereas permissionless blockchains depend on anonymous nodes to validate transactions. In digital health systems, permissionless blockchains have advantages in preventing powerful actors in making decisions that favour individual groups at the expense of others. Permissioned blockchains on the other hand are regulated by the authority of a specific ecosystem, usually a consortium, and present opportunities for creating interconnected systems. Such interconnected system could be designed by a consortium of healthcare providers, with patients' data belonging to the blockchain, and patients identified through unique ID providing both security and privacy of the patient. A patients' information sharing marketplace could prevent information blocking, and if cyber-attack results in information blocking, permissioned blockchains would present two unique advantages. Firstly, anyone in the medical consortium could check how and when information and transactions are happening. Secondly, blocking the information would change the hash on which the unique ID is based. Considering the above, the advantages for digital health systems are that patients can transmit personal records without the risk of tampering because blockchains are immutable and traceable. Apart from patients' security with blockchain permissions required for patient's medical data sharing, there are advantages in preventing fake or illegal sale of medication and with checking points of origin. ## Artificial intelligence for automatic diagnosis Artificial intelligence systems are already in use for automatic diagnosis for conditions unrelated to COVID-19 (e.g. Zhongshan Ophthalmic Eye Center, China), and abnormal respiratory pattern classifiers are already in development for large-scale screening of people infected with COVID-19 [bib_ref] Abnormal respiratory patterns classifier may contribute to large-scale screening of people infected..., Wang [/bib_ref]. As previously discussed, IoT connected thermal cameras could feed data into artificial intelligence systems for temperature screening in busy transport areas such as airports or train stations [bib_ref] Digital technology and COVID-19, Ting [/bib_ref]. Such real-time data streams can support and enhance pandemic-risk management, but only if open protocols are set for safe and seamless communication between devices [bib_ref] On the coronavirus (COVID-19) outbreak and the smart city network: universal data..., Allam [/bib_ref]. Unnecessary fragmentation of data restricts knowledge on trends and decisions on containment [bib_ref] On the coronavirus (COVID-19) outbreak and the smart city network: universal data..., Allam [/bib_ref]. ## Intelligent diagnosis, monitoring and supervision Intelligent-assisted diagnosis, monitoring and supervision can be conducted relatively simply, and lessons can be learned from existing live tracking systems.Online questionnaires can be uploaded into a real-time cloud database, and models can be updated based on the latest real case medical data to increase accuracy with results integrated in mobile applications automatically. Diagnoses can be grouped and automatically placed into 'confirmed, suspected or suspicious', while simultaneously, patients can be classified into 'mild, moderate, severe or critical pneumonia' categories [bib_ref] Chinese experts' consensus on the internet of things-aided diagnosis and treatment of..., Bai [/bib_ref]. The automatic patient classification may provide questionnaire on severity of infection, while the patient diagnosis grouping provides reassurances on the likelihood of infection. Such reassurances are aimed at reducing the pressure on medical professionals by limiting the need for in-person interaction. ## Tracking cases of prolonged isolation Existing systems (e.g. Gov.uk/NHS vulnerability register [bib_ref] Integration of cloud computing and internet of things: a survey, Botta [/bib_ref] can be used to create a mechanism for supermarkets to distinguish between customers in prolonged or age-related isolation and customers who are essential workers and those who are not a priority for home delivery services. Since this (online self-registration) mechanism already exists, preservation of privacy can be based on the existing system. This could be connected with a community-managed app linking volunteers [bib_ref] Smart cyber-physical systems: beyond usable security to security ergonomics by design, Craggs [/bib_ref] to those who are self-isolating and need help shopping, for example. ## Tracking high-risk cases Detection and mapping of high-risk groups that are not currently being treated by the UK's NHS could help identify the most in need of immediate support due to isolation measures. In addition to tracking, digital health apps need to focus on predicting high-risk cases. Predicting vulnerability and criticality in patients can be achieved using ML models perhaps with input from activity sensors. ## Mental health impact mitigation The COVID-19 pandemic could trigger new and sometimes unexpected mental health problems. Some mitigation activities could include free meals or relief on financial challenges or free showers to promote outdoor sports during work breaks. Information stations, like reception desks, could promote expression of concerns and keep workspaces safe while addressing staff concerns and anxieties. Social distancing will create a significant impact on mental health, and mental healthcare support needs to adapt to addressing not only the feelings of financial loss but also the feeling of emotional loss, the loss of self-worth, loss of motivation and a loss of meaning in daily life. [bib_ref] Security risk assessment in internet of things systems, Nurse [/bib_ref] ## Discussion on gaps in scientific research Our search of historic and recent scientific records on pandemics and new forms of data, on the Web of Science Core Collection, resulted in very limited records. In some cases, we found only a single record. It is unclear if this result is caused by lack of funding or lack of interest in the past on this research topic. Although we used all data records from the Web of Science Core Collection, there could be gaps in the data we are using. It could be too soon for publications with COVID-19 to have been published-and we should compare how different will it be in a year's time. We plan to rerun these queries again to see how the results have changes. In future studies, we should be focused on publication times, and if a vaccine is identified in the next few months or years, we should investigate how many papers have been published, comparable with the increase in COVID-19 activity. Future studies should also look at other factors of the virus in literature. In any case, we can only discuss the gaps and results from the data records we currently have. Here is a brief outline of the gaps in scientific research. Currently, there are only five scientific publications on the Web of Science Core Collection under the search on 'COVID-19 and big data', and only 59 on 'pandemics and big data', using historical records since 1900 (to 2020). Search results on 'big data' alone produced 88,410 records. There are 0 results on 'COVID-19 and time-stamped data' and on 'COVID-19 and genomics data'. The search of historic records, from 1900 to 2020, on 'pandemic and time-stamped data' resulted in only 3 records and on 'pandemic and genomics data' in 43 records. The search on 'COVID-19 and highdimensional data' resulted in 0 records, and the search of historic records on 'pandemic and high-dimensional data' resulted in only 2 records. Spatiotemporal data: currently, there is only one scientific publication on the Web of Science Core Collection under the search on 'COVID-19 and spatiotemporal data' and only 24 historic records since 1900 (to 2020) under the search on 'pandemic and spatiotemporal data'. Scientific research is needed on using spatiotemporal data in pandemic management.https://coronaboard.kr/en/ 36 https://www.gov.uk/coronavirus-extremely-vulnerable 37 https://covidmutualaid.org/ Discussion on research objectives and research findings from the review Following the example of previous studies [bib_ref] Early decrease in blood platelet count is associated with poor prognosis in..., Zhao [/bib_ref] [bib_ref] Covid-19 pandemic by the 'real-time' monitoring: the Tunisian case and lessons for..., Chaari [/bib_ref] related to predictive, preventive and personalized conclusions and expert recommendations, in this section, we review our research objectives and present our conclusions and expert recommendations. The first objective of the paper was to review, compare and critically assess predictive, preventive and personalized digital technology responses to the COVID-19 pandemic around the world, with the aim of identifying a digital approach to COVID-19 and future global pandemics. Despite applying statistical software to analyse all of the existing literature on the Web of Science and other databases, the conclusion of the study is that we are currently not in a position to critically assess this technology and the best approach varies by country as it will be a function of wealth, smart phone usage and culture. Our recommendation on culture is that when choosing solutions, we must consider that in some societies like the USA, people tend to make decisions based on the individual ('shelter in place'), while others like China or Taiwan are more about the whole ('social isolation'), with Europe in the middle. Mirroring this, there are differing attitudes towards ceding privacy to the state or networks of corporations mentioned in the results. The second objective and a point of interest in this paper was to review predictive, preventive and personalized interoperable digital healthcare solutions. The aim of this objective was to promote research that prevents failures from the past, where the separate design of digital health solutions has led to lack of interoperability. Hence, the review includes case studies that investigate different interoperable digital healthcare systems. A diverse set of non-contact digital healthcare technologies are investigated in case studies from different regions in the world. In our conclusion, we found no increase in personal data security risks from the integration of these digital technologies. What we did find was that some technologies have been used effectively in healthcare for decades (e.g. teleconferencing, mobile health, collaboration apps, virtual meetings, robotics, artificial intelligence), but we still failed in preventing the COVID-19 pandemic from spreading globally at a very fast rate. Among many questions emerging from this review, in our recommendation, the following are considered to be important: (1) How will we approach the problem of global healthcare strategies for predictive, preventive and personalized pandemic management? (2) How will we deal with the lack of adoption of the reviewed non-contact technologies? (3) How will we address the lack of digital investment and technological adaptation in predictive, preventive and personalized healthcare systems? (4) How will we handle the resistance to change? Apart from the multiple benefits identified for future global pandemic management, the review does not identify any new or unaccounted cyber risks from developing these interoperable digital healthcare solutions. The third objective of the paper was to determine if the proposed new predictive, preventive and personalized technologies and data policies for mass surveillance linking personal data from health professionals to governments would actually address the pandemic crisis. The finding from the review paper is that public response is limited by a mistrust about data privacy and that this differs across countries, depending on the chosen public communication strategy. Most countries' digital responses involve combinations of big data analytics, integration of national health insurance databases, tracing travel history from individual's location databases, code scanning and individual's online reporting. What is missing in the COVID-19 pandemic around the world is an integrated approach for digital healthcare management. In the review paper, we identified a plethora of tried and tested noncontact predictive, preventive and personalized digital healthcare concepts (e.g. mHealth, virtual clinics) that could help with controlling and preventing future pandemics. Mass surveillance linking personal data from health professionals should not be collected and used by governments but to the non-contact digital health technologies. The surveillance approach promoted by tech companies, where data is processed on individual devices and only anonymised data is shared with approval of the device owner, seems safer than some proposals for creating national databases. The concerns that big tech companies would abuse our personal data are in some cases exaggerated. Such personal data is already stored in our mobile devices and shared on social media sites, in mobile health apps, wearable devices or with our GP surgeries, in compliance with current data privacy regulations. Hence, focus should be placed on secure integration of this data, which is already shared, for the purposes of COVID-19 and future pandemics detection, monitoring and management. Such integration would create numerous benefits for the healthcare system, and we should anticipate an increased usage of these existing methods for sharing personal data. Many new users would be unfamiliar with how mobile data is analysed on our secured individual devices and how anonymised data is shared. This would firstly trigger fear of personal data abuse; secondly it would expose users unfamiliar with data privacy to agreeing for their data being shared without understanding the data privacy policies. Hence, in times when mass personal data surveillance is necessary, a mass information campaign should also be focused on individual understanding of these new surveillance systems. This could be an opportunity for governments to enable tech companies to learn from the COVID-19 and develop predictive, preventive and personalized tools that could prevent or minimize the effect of future pandemics. We should avoid mistakes from the past, when we did not prepare and learn lessons from previous pandemics, that are reviewed in this paper. # Conclusion The most visible narrative from this review paper is that global pandemics require global response, and the world has failed to learn from previous pandemics. While big tech companies (Google and Apple) are promoting a unified global approach, some governments are promoting regional approaches. While regional approaches can work in localized geographical areas, the abilities of developing countries to adopt similar regional systems are questionable. Most of the required predictive, preventive and personalized technologies (e.g. mobile phones, mobile apps, low-cost connected devices (IoT) and Internet connection) are present in developing counties. But the abilities of some developing countries to develop regional predictive, preventive and personalized pandemic monitoring systems are questionable. Especially when considering the required speed for building such systems and the need to operate at low latency, this leads to the inevitable conclusion that such systems should be global and in place ready to be activated in cases of pandemics. Adding to this, the review study concludes that such global systems for pandemic management should also be designed as interoperable and coupled with existing digital non-contact healthcare systems. To contribute to the design of such predictive, preventive and personalized interoperable and coupled digital noncontact healthcare systems, this review paper presents a survey of the Internet of Things and social machine literature as well as the overlap with the COVID-19 pandemic management. It continues with a survey of technology-driven mitigative measures and suggests some possible applications of technology in the COVID-19 pandemic management. The review applied statistical software to review and analyse existing databases and concluded that we still lack the data to critically access what the best approach is to integrate digital technologies in healthcare and pandemic management. The best approach probably varies by country as it will be a function of wealth, smart phone usage and culture. The review was enhanced with case studies on predictive, preventive and personalized interoperability of digital healthcare solutions and found that some technologies have been used effectively in healthcare for decades, and the risk of personal data exposure was not enhanced by these technologies. But these technologies failed in preventing the COVID-19 pandemic from spreading globally at a very fast rate. These technologies have the potential to prevent rapid spread of pandemics and even contain pandemics. Hence, future efforts should be placed on predictive, preventive and personalized pandemic management strategies and pandemic preparedness. The world has contained pandemics in the past. Many regions have failed to use the technologies described in this report, or in many cases even to learn from past successes, and have failed to manage this pandemic. This could change, and since the data is currently incomplete, we will run this analysis again in a year and see how it has changed and how it compares with previous pandemics. This study represents a snapshot in time and can be used in future studies for investigating different stages of the pandemic. Finally, emerging mass population surveillance systems should be built in a predictive, preventive and personalized interoperable system, to promote advancement of non-contact digital healthcare concepts (e.g. mHealth), that could help controlling and preventing future pandemics. To promote such an interoperable approach, the new mass population surveillance system cannot rely solely on technology and need to operate as social machines. Social machines with large numbers of human-computer participants can provide predictive, preventive and personalized answers to different and complex pandemic monitoring problems. [fig] 13: https://www.theguardian.com/commentisfree/2020/mar/20/south-korearapid-intrusive-measures-Covid-19#maincontent 14 https://www.smartcitiesworld.net/news/news/south-korea-to-step-uponline-coronavirus-tracking-5109 15 https://coronamap.site/ 16 https://coronaita.com/#/ in scientific research on pandemics. When we compare the 3708 historical records on global pandemics on the Web of Science Core Collection, with 372,454 results on business, 573,901 results on art and 455,356 records on fishing, then the problem becomes concerning. The world simply does not seem prepared for COVID-19, not to mention a deadlier global pandemic. Similar conclusions are presented in our review of new forms of digital data used in pandemic management. [/fig] [fig] Figure 2 19, Figure 3: Results of a statistical bibliometric analysis of the 500 most relevant research records on COVID-Corresponding author by country-COVID-19 research in 2020 [/fig] [fig] Figure 5: Collaboration network as a social structure of COVID-19 scientific research records in 2020 [/fig] [fig] Figure 4: Web of Science-results analysis tool management. [/fig] [fig] Figure 6: Historic analysis of scientific research on global pandemics 1900-2020 [/fig] [fig] Figure 7: Digital solutions for pandemic management [/fig] [fig] Figure 8: Blockchain in digital health systems integrated to COVID-19 virtual clinics (5) How do we stop the problem of fake news about COVID-19 (and future pandemics) and cyber risks in personal data sharing with these established and regulated systems. [/fig] [table] Table 1: Evolution of the Social Collaborative Internet of ThingsThe evoluƟon of Social CollaboraƟve Internet of Things [/table]

HMDB 3.0—The Human Metabolome Database in 2013 The Human Metabolome Database (HMDB) (www. hmdb.ca) is a resource dedicated to providing scientists with the most current and comprehensive coverage of the human metabolome. Since its first release in 2007, the HMDB has been used to facilitate research for nearly 1000 published studies in metabolomics, clinical biochemistry and systems biology. The most recent release of HMDB (version 3.0) has been significantly expanded and enhanced over the 2009 release (version 2.0). In particular, the number of annotated metabolite entries has grown from 6500 to more than 40 000 (a 600% increase). This enormous expansion is a result of the inclusion of both 'detected' metabolites (those with measured concentrations or experimental confirmation of their existence) and 'expected' metabolites (those for which biochemical pathways are known or human intake/exposure is frequent but the compound has yet to be detected in the body). The latest release also has greatly increased the number of metabolites with biofluid or tissue concentration data, the number of compounds with reference spectra and the number of data fields per entry. In addition to this expansion in data quantity, new database visualization tools and new data content have been added or enhanced. These include better spectral viewing tools, more powerful chemical substructure searches, an improved chemical taxonomy and better, more interactive pathway maps. # Introduction The human metabolome can be defined as the complete collection of small molecule metabolites found in the human body [bib_ref] Meet the human metabolome, Pearson [/bib_ref]. These small molecules include peptides, lipids, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, minerals, food additives, drugs, toxins, pollutants and just about any other chemical (with a molecular weight <2000 Da) that humans ingest, metabolize, catabolize or come into contact with. Together with the genome and the proteome, the human metabolome essentially defines who and what we are. However, in contrast to the genome and proteome, the metabolome itself is not easily defined. This is because the human metabolome is not solely dictated by our genes. Our environment (what we eat, breathe, drink) and our microflora (the bacteria that live in our intestinal tract) contribute to the metabolome. In other words, the human metabolome consists of a mix of both endogenous and exogenous compounds. Endogenous metabolites are small molecules synthesized by the enzymes encoded by our genome or our microfloral genomes, and exogenous metabolites are 'foreign' or xenobiotic chemicals consumed as foods, drinks, drugs or other 'consumables'. The fact that so many different chemicals from so many different sources can potentially appear in the human metabolome has made its characterization difficult. Nevertheless, several concerted efforts have been made to decipher the human metabolome-or, more appropriately, the human metabolomes. Beginning in 2005 and continuing to the present, the Human Metabolome Project (HMP) (2) has been using a variety of high-throughput metabolomic studies in combination with comprehensive literature surveys to compile as much information about the human metabolome(s) as possible. This information is released publicly through the Human Metabolome Database or HMDB [bib_ref] HMDB: a knowledgebase for the human metabolome, Wishart [/bib_ref]. First introduced in 2007, the HMDB is currently the world's largest and most comprehensive, organism-specific metabolomics database. It contains spectroscopic, quantitative, analytic and physiological information about human metabolites, their associated enzymes or transporters, their abundance and their disease-related properties. Since its initial release, the HMDB website has been accessed more than 5 million times and its associated papers cited nearly 1000 times. Feedback from users has led to many excellent suggestions on how to expand and enhance HMDB's offerings. Likewise, continuing advances in the field of metabolomics along with ongoing data collection and curation by the HMP team has led to a substantial expansion of the HMDB's content. Here, we wish to report on these developments as well as the many additions and improvements appearing in the latest release of the HMDB (version 3.0). ## Database enhancements Details regarding the HMDB's overall layout, navigation structure, data sources, curation protocols, data management system and quality assurance criteria have been described earlier [bib_ref] HMDB: a knowledgebase for the human metabolome, Wishart [/bib_ref]. These have largely remained the same for this release. Here, we shall focus primarily on describing the changes and improvements made to the HMDB since 2009. More specifically, we will describe the: (i) enhancements to the HMDB's content, completeness and coverage, (ii) improvements to the HMDB's interface and MetaboCard layout, (iii) improvements to its spectral databases and spectral viewing tools and (iv) improvements to the HMDB's data downloads, data formats and data structures. ## Expanded database content, completeness and coverage A detailed content comparison between the HMDB (release 1.0 and 2.0) versus the HMDB (release 3.0) is provided in [fig_ref] Table 1: Content comparison of HMDB 1 [/fig_ref]. As seen in this [fig_ref] Table 1: Content comparison of HMDB 1 [/fig_ref] , the latest release of the HMDB now has detailed information on >40 000 metabolites, representing an expansion of nearly 600% over what was previously contained in the database. This increase is primarily a result of the significant expansion of both 'detected' metabolites [separated into two categories: (i) detected and quantified and (ii) detected not quantified] and 'expected' metabolites (those for which biochemical pathways are known or human intake/exposure is frequent but the compound has yet to be detected in the body). In previous releases of HMDB, no clear distinction was made between these categories. In this release, the distinction is made quite explicitly using a 'status' data field that is now part of HMDB's new chemical ontology. This new chemical ontology also distinguishes the source (or probable source) of these compounds using the 'origin' data field. Using this data field (for both detected and expected metabolites), compounds are further classified as being endogenous, microbial, drug derived, a toxin/pollutant, food derived or different combinations of the above. Among the 'detected' metabolites, the number has grown from 4413 (in version 2.0) to 20 900 (in version 3.0), or roughly by 450%. In many cases, these additions do not represent the discovery of new compounds, but what is known about human food and drug consumption patterns, we have every reason to believe that these compounds exist in the 'collective' human metabolome (but not necessarily in every human metabolome) and that they could be or will eventually be detected in human biofluids and/or tissues. Second, we believe the HMDB needs to provide resources, particularly, for the mass spectrometry (MS)-based metabolomics and lipidomics communities, to assist with the putative identification of novel or 'yet-to-be-seen' compounds through mass matching. The HMDB is not the first or only metabolome database to include 'expected' compounds. Indeed, METLIN (5) has been including all possible di-and tripeptide combinations (8400 compounds) for many years, whereas LipidMaps (6) has included many 1000s of lipid species that have not yet been formally identified or measured. We chose not to include tripeptides in this release of HMDB as the experimental evidence for stable, long-lived tripeptides is not yet as strong as it is for stable, long-lived dipeptides. As a general rule, the HMDB's expected metabolites do not contain the level of detail found in the experimentally detected HMDB entries. On average, expected metabolites in the HMDB contain 30-50 completed data fields, whereas detected metabolites typically have 80-120 completed data fields. Furthermore, users can readily select which sets of compounds (detected and quantified, detected, not quantified and expected) they wish to browse or search against. Nevertheless, with this substantial growth of metabolites in the HMDB, the database now has many more compounds than most other commonly used metabolite/ metabolome databases [KEGG (7) = 16 843, HumanCyc (8) = 1321, BiGG (9) = 1509 or LipidMaps (6) = 37 127]. Furthermore, more than 25 000 compounds in the HMDB are (not yet) in other chemical databases, including PubChem (10) and ChEBI [bib_ref] Chemical entities of biological interest: an update, De Matos [/bib_ref]. In addition to substantially increasing the number of metabolite entries, we have also increased the completeness of the HMDB's annotations for hundreds of metabolites by adding many more detailed compound descriptions, including more compound synonyms (450% increase), more compounds with biofluid concentration or tissue location data (20% increase) and a greater number of compounds with synthesis records (20% increase). Furthermore, HMDB now includes data on metabolite transporters, channels, symporters in addition to the usual enzyme-specific data. This is especially true for the 'Detected' metabolites. Over the past 2 years, a significant effort has also been undertaken to upgrade the number and quality of reference nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) and gas chromatographymass spectrometry (GC-MS) spectra in the HMDB. In particular, hundreds of additional reference MS and NMR spectra were collected, assigned and/or annotated by the curation team, whereas hundreds of additional annotated/assigned MS and NMR reference spectra were obtained from the BioMagResBank (12), METLIN (5) and MassBank [bib_ref] MassBank: a public repository for sharing mass spectral data for life sciences, Horai [/bib_ref]. This effort has led to a 30-50% increase in the number of compound reference spectra in the HMDB. In the 2009 release of HMDB, we described the addition of 30 new pathway diagrams to the database which supplemented a number of KEGG pathway diagrams. Since then, these early pathway diagrams have been substantially upgraded in terms of quality, quantity and viewability, particularly through the inclusion of our own SMPDB pathways [bib_ref] SMPDB: the small molecule pathway database, Frolkis [/bib_ref]. With 442 hand-drawn, zoomable, fully hyperlinked human metabolic pathway maps now in the HMDB, the total number of pathway diagrams (including disease and metabolic signaling pathways) in the HMDB has increased by 800%. Unlike most online metabolic maps, these HMDB/ SMPDB pathway maps are quite specific to human metabolism and explicitly show the compound structures, protein quaternary structures, enzyme cofactors and subcellular compartments where specific reactions are known to take place. All chemical structures in these pathway maps are hyperlinked to HMDB MetaboCards and all enzymes are hyperlinked to UniProt data cards. By making use of the infrastructure originally built for the SMPDB, metabolite, protein and/or gene transcript concentrations may now be interactively mapped onto each pathway diagram. Furthermore, each pathway diagram in the HMDB is fully searchable (via PathSearch) and each pathway now has a short synoptic description. For a number of years the HMDB has been manually classifying all compounds in the HMDB into chemical 'kingdoms', 'classes' and 'families'. This chemical taxonomy has proven to be useful for many researchers, but it has also been quite challenging to maintain and we have found a number of inconsistencies. Furthermore, there are now a growing number of alternate chemical taxonomies being used by other databases, such as BioCyc/MetaCyc (15), ChEBI [bib_ref] Chemical entities of biological interest: an update, De Matos [/bib_ref] and LipidMaps [bib_ref] LIPID MAPS online tools for lipid research, Fahy [/bib_ref]. In an effort to both standardize our own chemical classification scheme and to integrate other chemical taxonomies/ontologies, we have completely redone the chemical taxonomy in HMDB. For the new version, a similar hierarchical classification system is still being used (kingdom, super class, class, subclass, other descriptors, substituents, direct parent), but the classification process is now fully automated (using thousands of structure rules), fully defined (with thousands of definitions) and fully self-consistent. The new taxonomy also uses a 'consensus' terminology or naming convention that makes compound classification more consistent and more easily mapped to other databases' classification schemes. Furthermore, under the field 'other descriptors' most of the known classification or taxonomic terms from other chemical databases, if available for that compound, are now included. A more detailed description of the chemical taxonomy software is being prepared and will be submitted for publication shortly. In addition to this chemical taxonomy, the HMDB also has a chemical ontology. This ontology includes status (detected or expected), origin (endogenous, microbial, drug, etc.), biofunction (energy, signalling, buffer, etc.), application (nutrient, industrial chemical, pharmaceutical, etc.) and cellular location (membrane, cytoplasm, extracellular). We are hopeful that this new and improved chemical taxonomy and ontology will help to provide a common language for large-scale mammalian metabolome comparisons. Thanks to the feedback provided by HMDB's user community, a number of new data fields have been added to each MetaboCard. For instance, HMDB 3.0 now provides an additional set of 10 computed/predicted property descriptors including (i) water solubility, (ii) LogP (two different algorithms), (iii) LogS, (iv) pKa, (v) hydrogen acceptor count, (vi) hydrogen donor count, (vii) polar surface area, (viii) rotatable bond count, (ix) refractivity and (x) polarizability. While more computed structure descriptors are certainly available, these represent the most frequently used descriptors and should allow HMDB users to perform much more detailed computed structure queries, analyses and comparisons. We believe that one of the most important and unique features of the HMDB is the information it contains on metabolite concentrations (normal and abnormal), disease associations and tissue locations. Relative to the previous release, the HMDB now has 20% more of these clinically or physiologically relevant details. Over the past 3 years, several comprehensive studies conducted by the HMP's metabolomic analysis team have led to the experimental validation and accurate concentration measurements of thousands of compounds in cerebrospinal fluid [bib_ref] Multi-platform characterization of the human cerebrospinal fluid metabolome: a comprehensive and quantitative..., Mandal [/bib_ref] , serum [bib_ref] The human serum metabolome, Psychogios [/bib_ref] and urine [bib_ref] Prediction of skeletal muscle and fat mass in patients with advanced cancer..., Stretch [/bib_ref]. These data have been supplemented with recently published metabolome-specific studies on saliva [bib_ref] Understanding the human salivary metabolome, Takeda [/bib_ref] , prostate [bib_ref] Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression, Sreekumar [/bib_ref] and other tissues/ biofluids. Disease association and abnormal concentrations have also been added via the HMP's literature curation team. These additions should make version 3.0 of the HMDB substantially more useful for clinical chemists and physicians. They should also aid many metabolomics researchers in clarifying what should (and what shouldn't) be routinely found in certain human biofluids or tissues. In addition to significantly expanding the data content in HMDB, a major effort has been directed at improving the quality of HMDB's existing data. Over the past 3 years, thousands of compound names, synonyms and descriptions were expanded or corrected. Likewise, hundreds of new compound synthesis and concentration references were collected, checked and validated. Similarly, extensive checks were performed on all of HMDB's small molecule structures to confirm that they exhibit the correct structure, chirality and stereochemistry. In particular, we developed a custom structure-checking program that used direct structure comparison (via a Mol file) of each of HMDB's structures against the corresponding structures in other databases (PubChem, ChEBI, ChemSpider, etc.). Any HMDB structure that did not match with the corresponding structure in one or more of these external databases was flagged. Each of these was assessed and/or corrected manually by a team of trained chemists. In many cases, the HMDB structure was correct and the external database structure was found to be in error; in other cases, the HMDB structure was determined to be in error and was subsequently corrected. In addition to these changes, a substantial effort has also been put into identifying and correcting minor structural, image format, naming, annotation and spectral assignment errors in the HMDB. While a number of internal checking and editing procedures are used by the HMDB curation team [see [bib_ref] HMDB: a knowledgebase for the human metabolome, Wishart [/bib_ref] for details], we are particularly grateful to external users who identified particularly subtle errors or offered suggestions to improve the data quality. Overall, we are quite confident that the quality of the data in HMDB (version 3.0) is much better than previous releases. ## User interface improvements Relative to previous releases, the user interface improvements for this year's release of the HMDB are relatively modest. The most obvious change is the restructuring of the MetaboCard into 14 distinctive categories or superfields with clearly demarcated titles [fig_ref] Figure 1: A screenshot montage of the different HMDB views showing the new layout... [/fig_ref]. These superfields include: (i) record information, (ii) metabolite identification, (iii) chemical taxonomy, (iv) chemical ontology, (v) physical properties, (vi) spectra, (vii) biological properties, (viii) normal concentrations, (ix) abnormal concentrations, (x) associated disorders, (xi) external links, (xii) references, (xiii) enzymes and (xiv) transporters. We believe this should make the data easier to view and browse. In addition, most of the detailed enzyme and transporter data that used to be displayed on the MetaboCard, is now in a separate ProteinCard which can be accessed by clicking the button under each enzyme or transporter name. To further improve the browsing in HMDB, we have also implemented sortable tables in the Metabolite Browse menu. Clicking on the up/down arrows in the table identifiers allows one to resort metabolites by HMDB ID (numerically), Name (alphabetically), International Union of Pure and Applied Chemists (IUPAC) Name (alphabetically), Formula (Alphanumerically) and Mass (Numerically), going from largest to smallest, from A-Z or vice versa. In addition to the usual browse options (metabolite, disease, pathway, biofluid, chemical class and metabolite library), we have also added a Protein Browse option to version 3.0. Protein Browse allows users to browse through, view or sort the list of human proteins, enzymes and transporters that are associated with each metabolite. Each protein is linked to a 'ProteinCard', which contains additional molecular, physiological or ontological data (30 data fields) about that protein as derived from a variety of databases or calculated from its sequence. Small improvements have also been made to HMDB's Chem Query tool. Chem Query is the HMDB's chemical structure search utility. It can be used to sketch (through ChemAxon's freely available chemical sketching applet) or paste a SMILES string of a query compound into the Chem Query window. Submitting the query launches a structure similarity search that looks for common substructures from the query compound that matches the HMDB's database of known or expected human compounds. Users can also select the type of search (exact or Tanimoto score) to be performed. High-scoring hits are presented in a tabular format with hyperlinks to the corresponding MetaboCards. The Chem Query tool allows users to quickly determine whether their compound of interest is a known human metabolite or chemically related to a known human metabolite. The same ChemAxon structure querying applet is also used with the 'Find Similar Structures' button located at the top of every MetaboCard. ## Enhancements to spectral databases and spectral searching In metabolomics, most compounds are identified via spectral comparisons against libraries of known compound spectra. Consequently, there is a critical need by many metabolomics researchers for comprehensive, publicly accessible libraries of reference compound spectra. As mentioned earlier, the number and type of reference spectra in release 3.0 is now much greater than in earlier releases. But so too is the utility of these spectra. Earlier versions of the HMDB's NMR and MS spectral assignments, peak lists or annotation files were kept in an unconventional format (for NMR) or not available for download (for MS). With a growing consensus on data exchange formats for MS and NMR spectra and improved methods becoming available for viewing spectra we have undertaken a substantive update to the HMDB's spectral resources. All NMR spectral assignments are now available for download in an NMR-STARformat. This format captures all relevant spectral features, spectral collection conditions, assignments and chemical structure information. As before, all NMR spectra continue to be available as raw (FID) files and as simple images (PNG format). In addition to these changes for HMDB's NMR spectra, nearly all MS/MS spectra in the HMDB are now available in mzML format [bib_ref] mzML: a single, unifying data format for mass spectrometer output, Deutsch [/bib_ref]. The mzML format is rapidly becoming the preferred format for MS data exchange as it robustly captures all relevant spectral features, MS spectral collection conditions and associated annotations. As before, all MS spectra in the HMDB are also available as simple images (PNG format) and as simple mass list files. Significant improvements have also been made for HMDB's spectral viewing and searching tools. In particular, users can now select between expected (33 000), detected (7000) or combined (40 000) metabolites for their MS search routines. A simple adduct calculator generates more than 25 possible adducts (containing Na+, K+, NH 4 + and dimer adducts) for three different modes (positive ion, negative ion, neutral) for each compound, creating an effective database of more than 1 million masses for high resolution parent ion matching. ## Improvements in hmdb's data formats and data structures While many visible 'front-end' enhancements have been implemented, HMDB's back-end has also been significantly enhanced. First, the HMDB information management system (MetaboLIMS) has been substantially rewritten, allowing the HMP curation team to perform a far more facile uploading and monitoring of newly entered data. Second, the HMDB user interface has been completely rewritten using Ruby-on-Rails (changed from the original Perl). Third, all the structure information in the HMDB has been consolidated into a central chemical structure database (MolDB) that now allows more rapid querying, retrieval, monitoring and updating of structural data. Fourth, all the spectral in formation in the HMDB has been consolidated into a central spectral structure database (SpecDB) that now allows much more rapid querying, retrieval, monitoring and updating of spectral data. Fifth, as mentioned earlier, all the downloadable spectral data have been converted into files that conform to standard exchange formats (NMR-STAR for NMR data and mzML for MS/MS data). Of course, all chemical structural data in the HMDB is already in standard SDF, PDB and MOL formats, while all sequence data is in a standard FASTA format. The adoption of data exchange standards for all spectral, structural and sequence data should make data dumps and data downloads much easier. Finally, most of the HMDB data has been converted to an easily parsed XML format. This should make data downloads of the annotated database and the development of data extraction routines much simpler and far faster for programmers and database developers. # Conclusion The HMDB is a comprehensive, web-accessible metabolomics database that brings together quantitative chemical, physical, clinical and biological data about all experimentally 'detected' and biologically 'expected' human metabolites. Over the past 3 years, a significant expansion to the content and a significant enhancement to the database's capabilities have taken place. Many of these content additions and content corrections are the result of continued experimental and literature mining efforts by the HMDB curatorial and analytical staff. Likewise, many of the graphical interface and spectral viewing/downloading improvements, which arose primarily from external user suggestions, are the result of significant programming efforts by the HMDB software development team. Overall, we believe these improvements should make the HMDB much more useful to a much wider collection of metabolomics and clinical researchers. This report is certainly not the final word on the human metabolome, nor is it the final version of the HMDB. What this particular release of the HMDB provides is a 'snapshot' of the human metabolome as of 1 January 2013. No doubt the size of the human metabolome will continue to grow as will the need for additional resources or additional data fields to handle the ever-expanding needs of the community that the HMDB serves. [fig] Figure 1: A screenshot montage of the different HMDB views showing the new layout of the MetaboCards and the new structure search tool. [/fig] [fig] FUNDING: Genome Alberta (a division of Genome Canada); Canadian Institutes of Health Research (CIHR); Alberta Ingenuity Centre for Machine Learning (AICML); Alberta Innovates BioSolutions (AIBS). Funding for open access charge: Canadian Institutes of Health Research. [/fig] [table] Table 1: Content comparison of HMDB 1.0 with HMDB 2.0 and HMDB 3.0 [/table]

Utility of 3-D printing for cardiac resynchronization device implantation in congenital heart disease # Introduction Three-dimensional (3D) printing is increasingly being used to visualize relationships of anatomical structures and is becoming more popular in planning cardiac procedures. [bib_ref] Cardiac 3D printing and its future directions, Vukicevic [/bib_ref] 3D printing of the coronary sinus (CS) and its branches has not previously been described. The value of 3D models could be particularly high in the planning of cardiac resynchronization therapy (CRT) device implantation where procedural difficulty is anticipated. CRT implantation can be challenging in patients with congenital heart disease and other structural heart abnormalities where the anatomical relationships are complex. A precise 3D model can be beneficial for planning CRT implants to clarify essential anatomical details such as size of the right atrium, angulation of the CS take-off, and location of CS branches and their relationship to the systemic ventricle. We describe the use of a 3D printed model to assist in CRT lead implantation in a case of congenitally corrected transposition of the great arteries (ccTGA). The accurate representation of the CS and its branches helped the operators preselect an appropriate target branch and specific equipment required. ## Case report A 76-year-old woman with ccTGA and a permanent pacemaker for complete heart block was referred for consideration for upgrade of her device to a cardiac resynchronization defibrillator. Her right ventricle (RV) functioned as the systemic ventricle and had deteriorated in function in the preceding few months to an ejection fraction of 30%-35%. Her functional status had also declined and was consistent with NYHA functional class 3. She experienced shortness of breath on minimal exertion. Her relevant medical history included atrial fibrillation, severe systemic atrioventricular valve dysfunction, severe biatrial enlargement, and hypertension. She had a single-chamber pacemaker implanted with a transvenous lead in the pulmonary ventricle for complete heart block 20 years prior with a ventricular pacing percentage of 90%. Following consideration and discussion with the patient and her family, a decision was made to implant a cardiac resynchronization defibrillator. Difficulty with implanting the left ventricular lead was anticipated owing to the patient's congenital heart disease. Specifically, adequate lead position and appropriate choice of target CS branch for the systemic ventricular lead were important. Therefore, a preoperative cardiac computed tomography (CT) was performed using a GE Lightspeed VCT 64-slice CT Scanner (GE, Boston, MA) with 0.625 mm slice thickness [fig_ref] Figure 1: Three-dimensional [/fig_ref]. As per the standard protocol at our institution, 105 mL of iodinated contrast agent (Omnipaque-350, GE, Boston, MA) was injected, at a flow rate of 4.5 mL/s, via a needle in the antecubital vein followed by a 20 mL saline bolus chaser. Data acquisition was electrocardiogram-gated throughout examination and tube current was as per routine cardiac CT protocol. A 3D mesh model was created to assist the operators in visualizing the 3D structure of the heart and the relationship of the coronary branches to the systemic ventricle. The CT Digital Imaging as Communication in Medicine (DICOM) files were imported into 3D Slicer version 4.10.2 (www.slicer.org). A region of interest was created around the heart. The heart model was segmented by using the semiautomated "grow from seeds" extension. Segmentation defects were corrected by modifying seeds and manual editing. The final model, including the CS, was made hollow with a shell thickness of 2 mm. The file was imported into Formlabs Preform software (Formlabs Inc, Somerville, MA). Layer thickness was set to 0.1 mm and the model was printed in gray resin [fig_ref] Figure 1: Three-dimensional [/fig_ref]. The 3D model of the patient's heart revealed a patent subclavian vein, a severely enlarged right atrium, and a large CS with no obstruction or angulation of the ostium. There was a single large lateral branch of the CS in addition to the middle cardiac vein. The lateral vein was identified as the target vein. It was possible to visualize on the model that the placement of a lead in this branch would provide a lateral position appropriate for resynchronization. The angle of this branch to the CS was approximately 130 degrees and therefore we predicted that a sub-selector at this angle would be needed during the case to engage this branch. The operators examined the 3D model preoperatively. The procedure was performed as per the department's standard approach. The existing pacing lead was capped. A new defibrillator lead was placed in the pulmonary ventricle. A deflectable sheath (Attain 6227; Medtronic, Minneapolis, MN) was used to engage the CS. A CS venogram was performed, confirming 1 suitable target branch, which corresponded to the 3D model [fig_ref] Figure 1: Three-dimensional [/fig_ref]. A 130-degree sub-selector sheath (Attain Select; Medtronic) was used to advance an angioplasty wire into the target branch. A quadripolar lead (Attain Performa; Medtronic) was advanced over the wire into position. Pacing thresholds were suitable and this was determined to be an acceptable final lead position. The total procedure time was 2 hours and 7 minutes and fluoroscopy time was 9 minutes and 40 seconds. # Discussion Three-dimensional imaging and printing has been used in preoperative planning in cardiac surgery as well other disciplines. [bib_ref] 3D-printed models for surgical planning in complex congenital heart diseases: a systematic..., Batteux [/bib_ref] It has previously been used to clarify relationships of intracardiac structures and is becoming more popular in planning procedures in congenital heart disease owing to the complex spatial relationships. [bib_ref] Cardiac 3D printing and its future directions, Vukicevic [/bib_ref] As far as we are aware, this technique has not previously been used to plan CRT implantation. CRT implants have a failure rate of up to 10% in the general population. [bib_ref] Cardiac-resynchronization therapy with or without an implantable defibrillator in advanced chronic heart..., Bristow [/bib_ref] There are no large studies assessing failure rate of CRT implant in congenital heart disease, but it is likely to be higher owing to the complex anatomy. Three-dimensional printing may be important in this population to help plan procedures. Clinical congestive heart failure due to systemic right ventricular failure occurs in .30% of patients with ccTGA by the age of 45 years. [bib_ref] Management of systemic right ventricular failure in patients with congenitally corrected transposition..., Filippov [/bib_ref] There has been a demonstrated association between pacemaker implantation and deterioration of systemic ventricular (RV) function and worsening systemic atrioventricular valve regurgitation in ccTGA. [bib_ref] Cardiac conduction system in congenitally corrected transposition of the great arteries and..., Baruteau [/bib_ref] The benefit of CRT in ccTGA has been established in a few small studies. One recent study reported on 53 patients with ccTGA who underwent pacemaker implantation. [bib_ref] Impact of pacing on systemic ventricular function in L-transposition of the great..., Hofferberth [/bib_ref] There was a lower rate of worsening RV function on follow-up in patients who received biventricular pacing compared with those with univentricular pacing (21% vs 52%, P 5 .043). Of the patients (n 5 53) that received univentricular pacing only, 26% were upgraded to biventricular pacemakers owing to ## Key teaching points Clinical congestive heart failure due to systemic right ventricular failure is common in patients with congenitally corrected transposition of the great arteries (ccTGA). The benefit of cardiac resynchronization therapy (CRT) in ccTGA has been established in a few small studies. The complex anatomical relationships in the ccTGA population and other congenital heart diseases can result in challenges for CRT lead implantation. Cardiac computed tomography images can be used to reconstruct and print an accurate patientspecific 3-dimensional heart model in cases of congenital heart disease. The coronary sinus ostium and branches can be precisely represented to assist in CRT implant preoperative planning. deteriorating systemic ventricular function. Half of these patients responded to this treatment with an improvement in ventricular function. The anatomical variation in the ccTGA population can result in challenges for lead implantation. The CS has been shown to develop with the morphologic atria in ccTGA while the venous branches develop with the morphologic ventricles. 7 Therefore, the CS commonly drains into the right atrium. The venous branches, which would usually be targeted for placement of systemic right ventricular lead, are often small and short. A study examining 56 pathologic cardiac specimens from patients with ccTGA found the CS to have a predictable ostial location and anatomical course in 88% of cases. [bib_ref] The cardiac veins in congenitally corrected transposition of the great arteries: delivery..., Bottega [/bib_ref] The abnormalities of the CS observed in this study included atretic ostia, abnormal ostial location, or multiple ostia. In the patients with CS abnormalities, 70% had Thebesian veins with ostia 1 mm that could be considered an alternative option for access to the coronary venous system. There was also extensive collateralization between the RV and LV noted, which could make pacing the RV via the middle cardiac vein or anterior intraventricular vein possible. In this case, we were able to obtain an accurate 3D model of the CS and its branches that correlated to the findings of intraoperative venogram. The 3D model allowed visualization of the region of the systemic ventricle that the lead would contact following placement in the target vein. Preoperative planning using the 3D model may have contributed to the shorter procedure duration, by providing knowledge of the CS branch anatomy and required equipment. Prior studies reported median procedure times in non-congenital heart disease patients to be approximately 170 minutes. [bib_ref] Cardiac-resynchronization therapy with or without an implantable defibrillator in advanced chronic heart..., Bristow [/bib_ref] This technique can be used to help with preoperative planning in future cases of CRT implantation where procedural difficulty is anticipated and can help predetermine target branch for appropriate lead positioning. # Conclusion We were able to use cardiac CT images to reconstruct and print an accurate patient-specific 3D heart model in a case of ccTGA. The CS ostium and branches were precisely represented to assist in CRT implant planning. The benefit conferred by the 3D model in this case was in preoperative determination of equipment required and selection of appropriate target branches of CS lead. The opportunity for the operators to study this model preoperatively may have also contributed to shorter fluoroscopy and procedure duration. [fig] Figure 1: Three-dimensional (3D) printed model of congenitally corrected transposition of the great arteries for preoperative planning in cardiac resynchronization therapy (CRT) device implantation. A: Computed tomography imaging of the coronary sinus (CS). The arrow indicates the target branch of the CS. The asterisk indicates the main CS body. B: 3D printed model of the heart. The white arrowhead indicates the target branch. C: Venogram during CRT implant procedure. The white arrowhead indicates the target branch. LA 5 left atrium; LV 5 morphologic left ventricle; RA 5 right atrium; RV 5 morphologic right ventricle. [/fig]

Coronavirus Pandemic: What Nuclear Medicine Departments Should Know ## Severe acute respiratory syndrome coronavirus 2 COVID-19 is caused by a novel b-coronavirus (1) that has been given the name ''severe acute respiratory syndrome coronavirus 2'' (SARS-CoV-2). It belongs to the Coronaviridae family and is an enveloped positive-strand RNA virus. Coronaviruses are named for the crownlike spikes on their surface. The most likely origin of the novel coronavirus is zoonotic, given it has a genome 96% identical to that of a severe acute respiratory syndrome-like coronavirus found in bats. The novel virus has been detected in respiratory, fecal, and blood specimens of infected patientsand is reported to remain viable as an aerosol for up to 3 h. There are reports that transmission can occur via ocular surfaces, as infected droplets and bodily fluids might contaminate the human conjunctival epithelium. The virus was reported to be found in upper respiratory samples 1-2 days before the onset of symptomsand is thought to be spread mainly via asymptomatic carriers. ## Nuclear medicine staff and patients In terms of personal protective equipment (PPE), the World Health Organization recommends taking contact and droplet precautions before entering the room of a suspected or confirmed COVID-19 patient. These include wearing disposable gloves to protect the hands; a clean, nonsterile, long-sleeved gown to protect clothing; medical masks to protect the nose and mouth; and eye protection such as goggles or a face shield. Respirators (e.g., N95) are recommended for aerosol-generating procedures. With the increasing number of cases and the shortage of testing kits for COVID-19, there should be greater emphasis on infection-control and social-distancing measures for both the public and staff members in the health-care environment. Effective and efficient use of both staff and equipment in nuclear medicine departments is crucial for patient care and workplace safety. Several national and international bodies have reported numerous measures that might be implemented nationally and regionally. However, the policies and their implementation will vary from region to region. Departments should be aware of their national or local hospital policies and follow them accordingly. Numerous articles about radiology procedures on COVID-19 patients have appeared, but there is limited advice and information related to nuclear medicine services. Compared with conventional radiological imaging, the requirements and logistics for nuclear medicine imaging are relatively complex, such as scheduling appointments, contacting patients, maintaining regulatory compliance, prioritizing procedures, limiting the duration of scans, and preventing infection. Our top priorities should be ensuring the personal wellness of our staff and providing sufficient training and staff coverage to manage patients with suspected or confirmed COVID-19. The team should be made aware that there are asymptomatic carriers of the virus, and a good contact history is of use. We should also ensure that, in the waiting areas, patients have access to alcohol gel, hand-washing facilities, tissue boxes, and masks. Nuclear medicine reception staff should self-protect and be vigilant at all times and encourage patients to self-declare if they or any family members have symptoms or have recently traveled from places affected by COVID-19. The staff should ask specific and direct questions such as about a history of fever, dry cough, dyspnea, and fatigue. Patients should be encouraged to follow basic hygiene practices. The patient waiting area should be large enough for patients to maintain distance while seated, or patient appointments should be scheduled so as to avoid having too many patients in the waiting area at a given time. In general, the nuclear medicine staff, which includes technologists, nurses, and health-care assistants, are at risk of exposure to COVID-19. Unlike radiologic procedures, nuclear medicine procedures require radiotracer injection, and contact between the staff and patient is essential. In most cases, nuclear medicine procedures are outpatient-based; under limited circumstances, they are inpatient-based. Inpatients will be a combination of oncology and nononcology patients. There is a probability that patients with COVID-19 may be asymptomatic at the time they are in the department for their scan. Furthermore, it is possible that not all inpatients have been tested for COVID-19 before they are sent to the department for their scan. These scenarios pose a risk for all staff, from the reception area to the scanning room. In general, most nuclear medicine scanners are not portable, unlike radiography or ultrasound devices; therefore, the need for patients to come to the department for their scan is inevitable. Consequently, we should have a stringent mechanism in place to protect our staff and patients, as well as a contingency plan if staff are temporarily absent because of illness or quarantine, which might affect regular work in the department. Under the current circumstances, most departments based in hospitals that are COVID-19 hubs are postponing routine elective scans while continuing to provide urgent nuclear medicine scans (e.g., PET/CT scans for oncology patients) (Tables 2-4). Given the widespread transmission and the increased risk of asymptomatic patients, staff should use PPE according to the local policy. The PPE items must be donned before entering the patient area, and the donning and doffing procedure should be performed correctly. In general, the team should minimize the number of staff in each clinical encounter to reduce unnecessary movement into and out of injection or scanning rooms, and staff should wear PPE while escorting patients. Airborne transmission of COVID-19 continues to be debated. There is an ongoing dilemma on whether to do ventilation-perfusion scans. It is reported that airborne viruses can spread in air-conditioning and ventilation systems. Medical procedures associated with the generation of aerosols, such as ventilation scans and oxygen supplementation, might carry an increased risk of transmission. Therefore, some have suggested stopping ventilation-perfusion scan services because the ventilation scan is aerosol-based. In addition, the use of perfusion-only scans is unlikely to be of any benefit if COVID-19 is suspected, as the COVID-19 response might alter the macroaggregated albumin distribution. Others have proposed several alternatives, such as performing only perfusion imaging in, for example, pregnant patients or performing perfusion SPECT or SPECT/CT. Overall, it depends on the local conditions. Decisions should be based on national or regional policies, and special precautions, especially for personnel conducting these tests, must be taken. A chest radiograph should be mandatory before a ventilation-perfusion scan. The current reports suggest that asymptomatic COVID-19 carriers may have positive chest radiography results after 14 d of quarantine, even with no reverse-transcription polymerase chain reaction testing for COVID-19. The chest radiography findings in COVID-19 patients are reported to frequently show bilateral lower-zone consolidation (peaking at 10-12 d from symptom onset). ## Radionuclide therapies Nuclear medicine departments perform various radionuclide therapies for both benign and malignant disease. The These are examples based on consensus only, and responsibility lies with each institution or hospital to ensure its written policy adheres to that outlined by national public health guidance in its respective country and hospital. hospital and department providing these services should have a practical and realistic solution. The multidisciplinary team (MDT) must make a pivotal decision to continue or stop therapy service temporarily. The radionuclide therapy service depends on multiple factors, such as whether the treatment is outpatient or inpatient, the availability of beds for inpatient-based treatments, regular supply and delivery of radiopharmaceuticals, the risk of a patient contracting COVID-19 during the hospital stay, the staff skill mix (in case therapy staff is infected with the virus and substitute staff must be enlisted), robust selection criteria, and treatment of elderly cancer patients with comorbidities. Finally, when patients are treated, they should additionally consent to the risk of COVID-19 during their stay in the hospital, and the need for radionuclide therapy should balance against the risk of contracting COVID-19. These are examples based on consensus only, and responsibility lies with each institution or hospital to ensure its written policy adheres to that outlined by national public health guidance in its respective country and hospital. FDG = fluorodeoxyglucose; PSMA = prostate-specific membrane antigen; PRRT = peptide receptor radionuclide therapy; DOPA = 3,4-dihydroxyphenylalnine; CABG = coronary artery bypass grafting; NaF = sodium fluoride. Referrals must be reviewed by nuclear medicine consultants or in multidisciplinary setting. These are examples based on consensus only, and responsibility lies with each institution or hospital to ensure its written policy adheres to that outlined by national public health guidance in its respective country and hospital. ## Social distancing Physical isolation or distancing of staff from one another is crucial to prevent transmission from asymptomatic carriers. In the scan-reporting rooms, it is suggested that the workstations be separated by at least 2 m or 6 ft. First, the department should consider providing alternative technologic solutions that allow remote or off-site work for nuclear medicine consultants and residents (e.g., reporting of scans and protocolling procedures). Second, multidisciplinary meetings or case discussions should be web-based or teleconferenced. Several departments have opted for flexible rotations or schedules, such as by working in small teams or by working for 1 wk on-site and then 1 wk remotely. Establishing a group email list or a social media group to keep in touch and communicate effectively is essential. ## Imaging equipment Local hospitals should have clear policies and procedures in place for nuclear medicine staff who image suspected or confirmed COVID-19 patients. The standard operating procedures of the department or health-care system should be updated regularly as evidence evolves. van Doremalen et al.have studied how long the virus survives in the air and on surfaces. They confirmed that the novel coronavirus remained active for 48-72 h on plastic and stainless steel surfaces, 24 h on cardboard, and 4 h on copper. However, these times will vary under real-world conditions and might depend on the temperature, humidity, ventilation, and amount of virus deposited. The most important COVID-19 factors related to nuclear medicine include clean imaging techniques and decontamination of imaging equipment (e.g., SPECT/CT and PET/ CT scanners), in addition to decontamination of any surface that may have come into contact with respiratory droplets. In general, after the patients are scanned, the scanner and room surface should be disinfected to prevent potential spread, and appropriate training of environmental maintenance staff is recommended. Public Health England has published guidance entitled, ''COVID-19: Cleaning in Non-Healthcare Settings''. The risk of infection depends on several factors, such as the type of surfaces contaminated, the amount of virus shed from the individual, the time the individual spent in the setting, and the time since the individual was last in the environment. All surfaces that the symptomatic person might have come into contact with must be cleaned and disinfected (e.g., visible body fluids, imaging equipment, chair, bathrooms, door handles, telephones, and grab-rails in corridors and stairwells). The PPE should be worn for cleaning an area where a person with possible or confirmed COVID-19 has been. Public Health England recommends the use of a combined detergent disinfectant solution at a dilution of 1,000 parts per million available chlorine; if an alternative disinfectant is used within the organization, this alternative should be checked to ensure that it is effective against enveloped viruses. The British Society of Thoracic Imaging has produced action cards to assist with designing local radiology standard operating procedures for patients who have or are at risk of COVID-19 (e.g., transferring a patient to a CT scanner or performing a CT scan). These action cards might be applied to nuclear medicine departments as well. However, they are examples only, and the responsibility lies with each institution or hospital to ensure that its written policy adheres to the national public health guidance in the respective country. ## Radiopharmaceuticals The functioning of nuclear medicine procedures depends on the availability of radioisotopes and kits. These are not always locally produced; nuclear medicine centers might have to rely on obtaining them from national or international supply-and-distribution channels. In the current scenario, with land and air traffic lockdowns, a shortage of radioisotopes and kits is expected, and it is difficult to predict when the shortage will occur or for how long. For efficient use of kits, block booking of specific procedures should be envisaged. Alternately, PET/CT scans can be used in place of single-isotope methods for some indications (e.g., bone imaging with 18 F-NaF and infection imaging with 18 F-FDG). Myocardial perfusion imaging can be performed as a 1-d protocol (stress-rest). Local radiopharmacists or managers of nuclear medicine departments should contact the suppliers and update the local team so that bookings can be planned accordingly. In comparison to SPECT services, PET centers with local cyclotrons might continue to function as usual in most cases. For departments without cyclotrons, the availability of 18 F-FDG will depend on local conditions. ## Staff well-being The current scenario might cause psychologic distress, social insecurity, and financial insecurity. Staff coming to work at the hospital are concerned that they might contract the virus and expose their friends or family. We should try to provide relevant and reliable information to allay their fears (e.g., social distancing, infection control, and self-quarantine). There should be specific local guidelines for viral testing of staff returning to work after illness. The team should remain connected with one another or with their friends and families by such means as group email, e-portals, and social media. Departments that are active in teaching and training can use online teaching material and webinars, which are available from most national and international nuclear medicine societies, as an alternative to face-to-face interaction and learning. National organizations should make some of their online education material available free to its members. Research work will be challenging during the current circumstances (except for research related to COVID-19), as most institutions have suspended their projects, and alternative ways of collaborating should be envisaged to prevent disruption of vital projects. ## Chest findings for pet/ct and spect/ct Incidental parenchymal lung abnormalities on chest images have been reported for patients with COVID-19, and prompt recognition may be useful for timely isolation and treatment (Figs. 2-5) (1). The chest radiographic and CT appearance of COVID-19 has been reported to overlap significantly with the findings for other types of coronavirus infections. Chest CT is reported to be an essential component in the diagnostic algorithm for patients with suspected COVID-19. The reported sensitivity of chest CT in detecting COVID-19 at the initial presentation is 56%-98% during the early stages of disease development, and the specificity is low (25%). Chest CT has limited sensitivity and a low negative predictive value early after symptom onset and is unlikely to be used as a reliable independent tool to rule out COVID-19. The initial findings in infected patients from Wuhan have shown bilateral lung opacities. The typical features include lobular and subsegmental areas of consolidation. Other groups have reported high rates of ground-glass opacities and consolidation, sometimes with a rounded morphology and peripheral lung distribution. The more extensive disease is reported to be seen on CT approximately 10 d after the onset of symptoms. The frequency of CT findings is related to the infection time course. On the basis of the current evidence, there are ground-glass abnormalities in the early disease phase, followed by crazy paving and increasing consolidation later in the disease course. Multifocal involvement is reported to be common, and the CT signs gradually improve approximately 14 d after symptom onset. The hallmark of COVID-19 on CT is ground-glass opacities and consolidation or pulmonary opacities (often with a bilateral and peripheral lung distribution) (31,32). Bernheim et al. have reported the absence of ancillary CT findings such as pleural effusions, lung cavitation, pulmonary nodules, and lymphadenopathy. Bai et al. have assessed the performance of U.S. and Chinese radiologists in differentiating COVID-19 from viral pneumonia on chest CT and found high specificity but moderate sensitivity. The British Society of Thoracic Imaging has published reporting guidance and a proforma document (which might help to report findings with speed and accuracy), as well as a teaching library. Its content will be accessible without a log-on via the British Society of Thoracic Imaging website. COVID-19-suspected pneumonia is 18 F-FDG-avid and might be detected as an incidental finding in asymptomatic patients undergoing PET/CT. The nuclear medicine community should be vigilant about looking for other unexpected scan findings that might reflect the effects of COVID-19 exposure or infection. COVID-19 has changed the way we work. We should stay informed, support each other, and provide practical solutions for safety and social well-being during these 3. Unenhanced axial CT image from 56-yearold woman shows groundglass opacities with rounded morphology (arrows) in right middle and lower lobes. Left lung was normal. (Reprinted with permission of (32).) ## Figure 4. Unenhanced axial CT image from 42-yearold man in late time group (10 d from symptom onset to this scan) shows bilateral consolidative opacities with striking peripheral distribution in right lower lobe (solid arrows) and with rounded morphology in left lower lobe (dashed arrow). (Reprinted with permission of (32).) uncertain times. We should adhere to our national and international recommendations. The health-care system and professionals must aim to deliver safe patient care, maintain a safe workplace, and ensure personal wellness. Life imposes things on you that you can't control, but you still have the choice of how you're going to live through this. -Celine Dion # Disclosure No potential conflict of interest relevant to this article was reported.

Cisplatin resistance-related multi-omics differences and the establishment of machine learning models Objectives: Platinum-based chemotherapies are currently the first-line treatment of non-small cell lung cancer. This study will improve our understanding of the causes of resistance to cisplatin, especially in lung adenocarcinoma (LUAD) and provide a reference for therapeutic decisions in clinical practice.Methods: Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and Zhongshan hospital affiliated to Fudan University (zs-cohort) were used to identify the multi-omics differences related to platinum chemotherapy. Cisplatin-resistant mRNA and miRNA models were constructed by Logistic regression, classification and regression tree and C4.5 decision tree classification algorithm with previous feature selection performed via least absolute shrinkage and selection operator (LASSO). qRT-PCR and western-blotting of A549 and H358 cells, as well as single-cell Seq data of tumor samples were applied to verify the tendency of certain genes.Results: 661 cell lines were divided into three groups according to the IC50 value of cisplatin, and the top 1/3 (220) with a small IC50 value were defined as the sensitive group while the last 1/3 (220) were enrolled in the insensitive group. TP53 was the most common mutation in the insensitive group, in contrast to TTN in the sensitive group. 1348 mRNA, 80 miRNA, and 15 metabolites were differentially expressed between 2 groups (P < 0.05). According to the LASSO penalized logistic modeling, 6 of the 1348 mRNAs, FOXA2, BATF3, SIX1, HOXA1, ZBTB38, IRF5, were selected as the associated features with cisplatin resistance and for the contribution of predictive mRNA model (all of adjusted P-values < 0.001). Three of 6 (BATF3, IRF5, ZBTB38) genes were finally verified in cell level and patients in zs-cohort.Conclusions: Somatic mutations, mRNA expressions, miRNA expressions, metabolites and methylation were related to the resistance of cisplatin. The models we created could help in the prediction of the reaction and prognosis of patients given platinum-based chemotherapies.In this study, we aim to find out the causes of resistance to cisplatin from the genetic, pharmacological, and cellular level as well as the prognosis of patients who have undergone platinum-based chemotherapy, and provide a reference for therapeutic decisions in clinical treatments. # Introduction Cisplatin was first synthesized by M. Peyrone in 1844 and its chemical structure was first elucidated by Alfred Werner in 1893. However, the compound did not gain sufficient scientific investigations until the 1960's, when Rosenberg found that it was capable of inhibiting cell division in Escherichia coli, which increase the possibility of its use in cancer chemotherapy [bib_ref] Cisplatin in cancer therapy: molecular mechanisms of action, Dasari [/bib_ref] [bib_ref] Inhibition of cell division in Escherichia coli by electrolysis products from a..., Rosenberg [/bib_ref]. In 1978, cisplatin became the first FDA-approved platinum compound for cancer treatment [bib_ref] The resurgence of platinum-based cancer chemotherapy, Kelland [/bib_ref] , and later it became one of the most important anticancer drugs. Nowadays, platinum-based chemotherapy remains an important treatment modality for these patients with advanced NSCLC due to the emergence of resistance to targeted therapies of EGFR, ALK, or ROS mutant tumors [bib_ref] Platinum-based chemotherapy in advanced nonsmall-cell lung cancer: optimal number of treatment cycles, Rossi [/bib_ref]. For the mechanism of its pharmacology, generally, they damage DNA, leading to cell cycle arrest and cell death, typically via apoptosis [bib_ref] The role of apoptosis in cell killing by cisplatin: a flow cytometric..., Ormerod [/bib_ref] [bib_ref] Cisplatin: mode of cytotoxic action and molecular basis of resistance, Siddik [/bib_ref]. However, side effects and drug resistance are the two inherent challenges of cisplatin that limit its application and effectiveness [bib_ref] The first metal based anticancer drug, Ghosh [/bib_ref]. In many common tumor types such as NSCLC, the therapeutic efficacy of platinum-based DNA damaging agents is limited, resulting in only about one-third of patients receive benefits [bib_ref] Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair..., Kong [/bib_ref] [bib_ref] Early response to platinum-based first-line chemotherapy in non-small cell lung cancer may..., Sirohi [/bib_ref]. By now, at least four distinct classes of mechanisms by which cancer cells become resistant to cisplatin-based chemotherapy have been developed and targeting at least two distinct mechanisms might be the most successful strategies for circumventing resistance [bib_ref] Molecular mechanisms of cisplatin resistance, Galluzzi [/bib_ref]. In this study, we aim to find out the causes of resistance to cisplatin from the genetic, pharmacological, and cellular level as well as the prognosis of patients who have undergone platinum-based chemotherapy, and provide a reference for therapeutic decisions in clinical treatments. # Methods ## Data processing Details of cell lines information were downloaded from Cancer Dependency Map (Depmap, depmap.org) and Cancer Cell Line Encyclopedia (CCLE, https:// porta ls. broad insti tute. org/ ccle/ data), including IC50 value, cell line source, somatic mutation, mRNA expression, miRNA expression, and metabolite. The information of lung adenocarcinoma patients treated with cisplatin was downloaded from The Cancer Genome Atlas (TCGA, https:// gdc. cancer. gov/) (TCGA-LUAD) with their gene expression data. As the CCLE, Depmap, TCGA databases are open to the public under specific guidelines, it confirms that all written informed consents were obtained before data collection. # Differential analysis Differential analysis of somatic mutation, RNA, miRNA, and metabolite data between low IC50 and high IC50 groups was performed with R (version 3.6.1). Maftools, the R package, was used to summarize, analyze and visualize the somatic mutation data. RNA, miRNA, and metabolite data were first normalized and standardized by constructing relevant expression matrices using edgfR after removing those without enough sequence fragments in the sample. Differential genes, somatic mutations, miRNAs (P < 0.05, false discovery rate (FDR) < 0.05) were sorted according to logFoldChange values (|logFC|> 1) to identify significantly different expressions. All the differential analyses were presented in a heat map and volcano plots. ## Go, kegg and gsva analyses GO analysis and GSVA analysis were performed to investigate the biological implications of proteins significantly associated with platinum response. R (version 3.6.1) was used for GSVA as well as GO and KEGG pathway enrichment analyses. The significance level was set to 0.05 for the corrected P-values. Bar map and dot map were used to visualize the consequences. # Model contribution The characteristic of LASSO regression is to consider both Variable Selection and Regularization when fitting a generalized linear model, for applying which, the "Glmnet" (Lasso and Elastic-Net Regularized Generalized Linear Models) R package via penalized maximum likelihood fitness was used, and the mRNAs and miRNAs mostly relative to the resistance to cisplatin were obtained. Logistic regression, classification and regression tree (CART), and C4.5 decision tree classification algorithm, which use occurrence ratio to determine the category of the dependent variable, was based on the results of LASSO. Finally, we use stepwise regression to select variables, and contribute the predictable model. ## Cell culture and cytotoxic assay NSCLC A549 and H358 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were fostered in RPMI-1640 containing 10% fetal bovine serum (FBS) and 100 μg/mL of penicillin-streptomycin with or without DDP (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) added into the culture medium for incubation in a humid atmosphere containing 5% CO 2 at 37 °C. Cell proliferation was evaluated by Cell Counting Kit-8(CCK-8; Dojindo, Kumamoto, Japan). Briefly, 2 × 10 3 of A549 and H358 cells were plated in 96 well plates. They were incubated with 100 μL RPMI-1640 containing 10% fetal bovine serum (FBS) and 100 μg/mL of penicillinstreptomycin for 24 h, then with or without DDP (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for another 24 h at 37 °C. After treatment, cells were incubated in 10% CCK-8 reagent. The OD value was measured after 2 h at 450 nm with a microplate reader from Bio-Rad (Microplate reader 3550-UV). ## Rna interference siRNAs targeting BATF3, IRF5, ZBTB38, and Silencer Negative Control siRNAs were purchased from Ribobio (sequences provided in Additional file 6: Table S1). We purchased two different siRNAs for each gene to avoid the off-target effects. A549 and H358 cells were seeded in 6-well plates for 24 h prior to transfection with siRNA targeting BATF3, IRF5, ZBTB38, and corresponding non-targeting controls. A total of 150 nM of siRNA was added to each experiment made up of the target siRNA and topped up with the appropriate concentration of non-targeted controls where appropriate. Transfections were carried out in OptiMem medium (Gibco) using Lipofectamine 8000 transfection reagent (Beyotime). 48 h post-transfection, cells were harvested and assayed for RNA and protein expression levels of the target of interest. At the same time, corresponding samples were treated as described in the text. ## Rna preparation and qrt-pcr analysis To detect the expression of BATF38, IRF5, ZBTB38 in A549 and H358 cell lines, RT-qPCR was carried out on an QuantStudio ® 5 real-time PCR system (Applied Biosystems) with proper PCR parameters. Total RNAs were extracted by TRIzol (TIANGEN, Beijing, China). The first-strand cDNA was synthesized using Hifair ® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Tokyo, Japan) according to the manufacturer's instructions. Then Hieff ® qPCR SYBR Green Master Mix (Low Rox Plus) (YEASEN) was used with the following PCR parameters, 1 cycle of 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 34 s at 60 °C. β-actin was used as the reference. Primers used in this study are listed in Additional file 7: . All the samples were repeated three times. # Western blot analysis Proteins of A549 cells and H358 cells after RNA interference were extracted using RIPA (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktail (Topscience). Then these proteins were quantified by Enhanced BCA Protein Assay Kit (Beyotime). Proteins were then resolved, separated, and finally transferred into PVDF membranes under the influence of an electric current in a procedure (Merck-Millipore, Burlington, MA, USA). Membranes were blocked, followed by incubation with specific primary antibodies [bib_ref] Knockdown of SMAD3 inhibits the growth and enhances the radiosensitivity of lung..., Niu [/bib_ref]. Finally, we observed the protein bands by Moon chemiluminescence kit (Beyotime). The following antibodies were used: Rabbit anti-BATF3 (NBP2-41296, dilution 1:1,500, Novus Biologicals); Rabbit anti-IRF5 (CY5822, dilution 1:1,500, Abways); rabbit anti-ZBTB38 (21906-1-AP, dilution 1: 1,500, Proteintech), mouse β-ACTIN (1:3,000, AA128, Beyotime), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) (1:3,000, A0208, Beyotime), and HRP-labeled goat anti-mouse IgG (H + L) (1:3,000, A0208, Beyotime). ## Single tumor and immune cells We used the same methods described in our previous studies to test cisplatin-sensitivity-related genes [bib_ref] Landscape and dynamics of single tumor and immune cells in early and..., Chen [/bib_ref]. This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (B2019-137R). Patients had signed the informed consent at hospitalization. # Results ## Group division and overview of cells' ic50 value of cisplatin 661 cell lines were finally enrolled with the complete data of mRNA, microRNA expression, and metabolite. They were divided into 3 groups according to the IC50 value, the half inhibitory concentration, given by the CCLE database, which reflects the sensitivity to chemotherapeutic drugs. The lower the value, the stronger the sensitivity. In this study, the top 1/3 (220) with a low IC50 value (IC50 ≤ 10.4) were defined as the sensitive group, while the last 1/3 (220) with a low IC50 value (IC50 ≥ 26.2) were enrolled in the insensitive group . Obviously, the resistance to cisplatin varies among different cancers. In the Depmap database, we successfully classify different tumor cell lines according to their source of cancer, so as to obtain the average IC50 value of cisplatin of related cancers. As shown in Additional file 1: , the average IC50 value of 6 cancers were more than 100, including Lung adenocarcinoma (LUAD), Mesothelioma (MESO), Thyroid carcinoma (THCA), etc., which means patients with these types of cancer may suffer a higher possibility of resistance to cisplatin, one of the most common chemotherapy drugs. ## Somatic mutation difference The overall pattern of somatic mutation of the cell line in detail was described in Additional file 2: . After matching the somatic mutation data with the drug sensitivity data, the differences in the characteristics of somatic mutation were investigated between the low IC50 (218 in 220) and high IC 50 (220 in 220) groups. As shown in and B, although two somatic mutation oncoplots and co-occurring sets of these genes are similar to a certain extent, it is not difficult to find that in the cisplatin-resistant group (high IC50), the mutation of TP53 ranked the first (10%), which is higher than the mutation rate in the cisplatin-sensitive group (low IC50 group). As previously reported by Aditya Bagrodia [bib_ref] Genetic determinants of cisplatin resistance in patients with advanced germ cell tumors, Bagrodia [/bib_ref] , TP53 mutations are only found in cisplatin-resistant tumors, especially in primary mediastinal non-seminoma. Therefore, we might conclude that TP53 mutation is closely related to cisplatin resistance. In contrast, MUT5 had a higher mutation rate in the low IC50 group. We also checked for drug-gene interactions compiled from Drug Gene Interaction database with "drugInteractions" function. Most drugs are related to TTN and TP53, which is less different between the two groups of cells. ## Differential analysis from different levels gene functional analysis To further discover the differences between the 2 groups, differential analysis between the two cell lines was applied from different levels. First, at the mRNA level, through edgfR, we found 1348 mRNA were differentially expressed between two groups (P < 0.01), including 1 downregulating in the cisplatin resistance group (SLFN1) and 35 upregulating (TMEM4SB, CLDN3, KLK6, IRF5, ZBTB38, etc.) with an obvious fold change (|logFC| > 1) . Similarly, at the miRNA level, as shown in , 80 differentially expressed miRNA were discovered (P < 0.01) and 11 downregulating (miR-194, miR-206, miR-215, etc.,), 12 upregulating (miR-144, miR-16, miR-129-3p, etc.,) with an obvious fold change (|logFC| > 1). However, for the metabolizes, only the expression of 15 metabolites, such as urail, serine, carnosine, N-carbamoyl-β-alanine, cystathionine, etc., showed significant differences between the two groups, with no obvious fold change (|logFC| < 1) . For the methylation, none of the differential methylation sites with obvious fold change (|logFC| > 1) were closely related to the differentially expressed genes . We firstly performed a Gene Ontology (GO) analysis of the 1348 mRNAs that were found differentially expressed before based on the CCLE data. Gene functional enrichment analysis showed that 20 GO functional groups exhibited significant differences between the low IC50 group and the high IC 50 group, including actin-binding, extracellular matrix structural constituent, GTPase binding, guanyl-nucleotide exchange factor activity, etc. [fig_ref] Figure 5: K-M plot showing the differences of survival time between high and low... [/fig_ref]. For Kyoto Encyclopedia of Genes and Genomes (KEGG) based on these mRNAs [fig_ref] Figure 5: K-M plot showing the differences of survival time between high and low... [/fig_ref] , 10 pathways differed between two groups: PI3K-Akt signaling pathway, which had the most significant difference and the highest gene ratio, followed by proteoglycans in cancer, focal adhesion, etc. Next, to further conclude the difference of pathways and functions, we applied the Gene set variation analysis (GSVA), which takes specific gene sets as a characteristic expression matrix and quantifies the results of gene enrichment [fig_ref] Figure 5: K-M plot showing the differences of survival time between high and low... [/fig_ref]. In all, we concluded 643 gene pathways, most of which were related to drug resistance, including Riggins tamoxifen resistance, EGFR signaling 24HR, gefitinib resistance, KIM-MIC amplification targets, etc. ## Model construction and evaluation Firstly, the glmnet R package was applied with "family = binomial", which is suitable for binary discrete dependent variables to determine whether the DEGs were related to the high or low IC 50. 6 of the 1348 mRNAs, FOXA2, BATF3, SIX1, HOXA1, ZBTB38, IRF5, selected as the associated features with cisplatin resistance [fig_ref] Figure 4: cisplatin-sensitivity related model [/fig_ref] , and then related logistic model was established through glm [bib_ref] Regularization paths for generalized linear models via coordinate descent, Friedman [/bib_ref] function [fig_ref] Figure 4: cisplatin-sensitivity related model [/fig_ref]. Next, we applied J48 and rpart R package with " "fo rmu l a = ic 50 ~ FO XA2 + B ATF3 + SIX1 + HOXA1 + ZBTB3 8 + IRF5"" and " "fa mil y = gaussian"", which is sui tab le fo r c ontinuous univariate as w e a ime d to get the predicting value of IC50 value through this model (Additional file 3: . The step function is used to achieve stepwise regression. From the results, it is found that 3 in 6 variables had passed the significance test (p < 0.05) and became relatively important variables for constructing the model. While each variable in the model passed the significance test, it is necessary to ensure that the entire model is significant. Therefore, the Chi-Squared test was performed on the model. As the variables were added to the model one by one from the first to the last, the model finally passed the significance test, indicating that the model composed [fig_ref] Table 1: Coefficient display of logistics regression equation model for miRNA [/fig_ref] of these variables is meaningful and correct. Finally, we testified whether these genes are not only related to cisplatin resistance, but also have connection with patient survival. In addition, according to the data in the TCGA database, their expression levels were related to the prognosis of different tumor patients, including breast cancer, ovarian cancer, lung cancer and gastric cancer to a certain extent [fig_ref] Figure 5: K-M plot showing the differences of survival time between high and low... [/fig_ref]. Indeed, BATF3, IRF5 and ZBTB38 also contributed in evaluation of the response to chemotherapy of breast and ovarian cancer patients (Additional file 4: [fig_ref] Figure 4: cisplatin-sensitivity related model [/fig_ref]. Similarly, we composed the miRNA predicting model. From the LASSO regression, 4 significant miRNAs were concluded, miR-203, miR-200c, miR-148a, miR-142-5p (Additional file 5: [fig_ref] Figure 5: K-M plot showing the differences of survival time between high and low... [/fig_ref]. After the step function, miR-203 and miR-200c left, both of which passed the significance test conducted by Chi-Squared test, indicating that the model composed [fig_ref] Table 1: Coefficient display of logistics regression equation model for miRNA [/fig_ref] of these variables is meaningful and correct. ## Expression of batf3, irf5, zbtb38 is associated with the sensitivity of cisplatin in a549 and h358 cells and tumor samples To test the causal relationship between BATF3, IRF5, ZBTB38 expression and sensitivity to cisplatin on the cell level, we transfected BATF3-targeting, IRF5-targeting, and ZBTB38-targeting siRNAs separately in human cancer cell lines, A549 and H358 (non-small-cell lung cancer), which on the one hand expresses high BATF3, IRF5, ZBTB38 levels. These two cell lines in the result of previous sequencing analysis, and on the other hand, were constantly used in our lab. All siRNA-mediated silencings proved to be highly efficient and were sustained for 3d or more. IRF5 and ZBTB38-silenced cells exhibited at least a reduced sensitivity to cisplatin compared with mocksilenced cells, while the BATF3-silenced cells exhibited increased sensitivity to cisplatin (P ≤ 0.01, [fig_ref] Figure 6: Silencing BATF3 significantly increases sensitivity to cisplatin, while silencing IRF5, ZBTB38 reduces [/fig_ref]. A total of 6 ESCC (Esophageal Squamous Cell Carcinoma) patients were selected, who accepted complete cisplatin-containing neoadjuvant chemotherapy treatment, including 4 neoadjuvant-chemotherapy insensitive (NACT-NON-SEN) tumor samples and 2 neoadjuvantchemotherapy sensitive (NACT-SEN, complete response [CRs]) tumor samples [fig_ref] Figure 7: tSNE of tumor cells, BATF3, IRF5, ZBTB38 in NACT-NON-SEN and NACT-SEN groups... [/fig_ref]. RECIST standard was used to evaluate the effectivity of NACT effect. Similarly, 5 LUAD patients were selected, 3 of which were NACT-SEN (3 CRs, 1 PRs) and 2 were NACT-NON-SEN [fig_ref] Figure 7: tSNE of tumor cells, BATF3, IRF5, ZBTB38 in NACT-NON-SEN and NACT-SEN groups... [/fig_ref]. In LUAD, BATF3 significantly down-regulated in the NACT-SEN group, while the expression of IRF5, ZBTB38 in the NACT-SEN group were significantly higher than the NACT-NON-SEN group in malignant epithelial cell cluster (marked with EPCAM and SOX4, [fig_ref] Figure 7: tSNE of tumor cells, BATF3, IRF5, ZBTB38 in NACT-NON-SEN and NACT-SEN groups... [/fig_ref]. In ESCC, BATF3, IRF5, ZBTB38 showed the same tendency in the 9801 malignant cells marked by LYZ and C1QB [fig_ref] Figure 7: tSNE of tumor cells, BATF3, IRF5, ZBTB38 in NACT-NON-SEN and NACT-SEN groups... [/fig_ref]. # Discussion Cisplatin is one of the most potent and widely used drugs for the treatment of various solid cancers such as testicular, lung, cervical cancer, etc., [bib_ref] The first metal based anticancer drug, Ghosh [/bib_ref] , while tumor responses to cisplatin or carboplatin depend on the levels of platinum-DNA adducts and the DNA repair capacity of the cells [bib_ref] The resurgence of platinum-based cancer chemotherapy, Kelland [/bib_ref] [bib_ref] Nucleotide excision repair: why is it not used to predict response to..., Bowden [/bib_ref]. The development of cisplatin resistance in human cancer cells, including cell growth-promoting, apoptosis, DNA damage repair, and endocytosis, all of which are mechanisms supporting cell survival [bib_ref] Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic..., Shen [/bib_ref]. In this study, we began with exploring the important genes and miRNA of different cancer cell lines related to the IC50 value of cisplatin, based on which, we constructed the predicting model of the cisplatin resistance using the expressing value of 6 mRNAs, 3 of which, BATF3, IRF5, ZBTB38 were also verified in our own samples. All of them are closely related to the immune. On the one hand, several studies reported that chemotherapy was able to modulate the function of TAM [bib_ref] Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or..., Caro [/bib_ref] [bib_ref] Reduction of gender-associated M2-like tumor-associated macrophages in the tumor microenvironment of patients..., Matsuki [/bib_ref] [bib_ref] Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive..., Pinto [/bib_ref]. On the other hand, the different alteration of TAM reflects patients' responses to chemotherapy [bib_ref] The Role of tumor associated macrophages (TAMs) in cancer progression, chemoresistance, angiogenesis..., Dallavalasa [/bib_ref] [bib_ref] Tumor microenvironment in chemoresistance, metastasis and immunotherapy of pancreatic cancer, Wang [/bib_ref] [bib_ref] Neoadjuvant chemotherapy induces IL34 signaling and promotes chemoresistance via tumor-associated macrophage polarization..., Nakajima [/bib_ref] [bib_ref] The microenvironment of lung cancer and therapeutic implications, Mittal [/bib_ref]. Coincidentally, the three genes we selected are all related to immunity, which might suggest that they affect tumor cell sensitivity to cisplatin by regulating TAM. The interaction between BATFs and IRFs in immune cell lineages occurs in the gene expression network of several crucial processes. For example, BATF and IRF4 cooperate in CSR, as well as in antibody class switching through influencing T FH cells and germinal center B cells [bib_ref] Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks, Murphy [/bib_ref]. What's more, the cooperation of ZBTB46, BATF3, and IRF8 activates the development of CD8α+ conventional dendritic cells (cDCs) [bib_ref] Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC..., Grajales-Reyes [/bib_ref]. In this study, we found ZBTB38, BATF3, and IRF5 might have interactions that affect the cell sensitivity to cisplatin, but with various trends. Basic leucine zipper transcription factor ATF-like (BATF), BATF2, and BATF3 belong to the activator protein 1 (AP-1) family of transcription factors, which regulate numerous cellular processes [bib_ref] The Pfam protein families database, Finn [/bib_ref]. BATF3 was first identified in human T cells and was later found to play a critical role in the development of the cDC1 subset of conventional DCs [bib_ref] Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC..., Grajales-Reyes [/bib_ref] [bib_ref] Compensatory dendritic cell development mediated by BATF-IRF interactions, Tussiwand [/bib_ref]. Although we found that knocking down the expression of BATF3 will increase cancer's sensitivity to cisplatin, the main function of this transcription factor is to activate CD8alpha+ dendritic cells. In other words, it plays important role in cross-presentation in tumor rejection and deletion of the transcription factor Batf3 ablated development of CD8alpha + dendritic cells [bib_ref] Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic..., Hildner [/bib_ref]. Furthermore, within the OpACIN trial, severe melanoma patients suffering from the reoccurrence of tumor after adjuvant or neoadjuvant consisting ipilimumab + nivolumab displayed a low level of Batf3 + DC-associated genes [bib_ref] Batf3(+) DCs and type I IFN are critical for the efficacy of..., Liu [/bib_ref] , which might reveal the two-side adjusting effects of BATF3 on chemotherapy and immunotherapy. Interferon regulatory factor-5 (IRF5) is a transcription factor and has essential cellular mechanisms as a tumor suppressor gene [bib_ref] Acetylation of interferon regulatory factor-5 suppresses androgen receptor and downregulates expression of..., Acidereli [/bib_ref]. The beneficial effects of NACRT on TAMs' infiltration might be associated with genderdependent IRF-5 expression, as CD163 + TAMs, which were related to poor prognosis [bib_ref] Significance of M2-polarized tumor-associated macrophage in pancreatic cancer, Kurahara [/bib_ref] , were shown to be negatively correlated with the number of IRF-5 + cells [bib_ref] The Role of tumor associated macrophages (TAMs) in cancer progression, chemoresistance, angiogenesis..., Dallavalasa [/bib_ref]. It was also reported that increased expression of IRF-5 in M2-like TAM promoted antitumor immune response to NACT [bib_ref] Significance of M2-polarized tumor-associated macrophage in pancreatic cancer, Kurahara [/bib_ref]. In our research, IRF-5 showed the same tendency as the knocking down of it could increase tumor cell's resistance to cisplatin with a higher IC50, and the potential mechanism might lie on its function of secretin IFN-α, because the delivery of IRF5 protein into human primary pDCs increased IFN-α secretion [bib_ref] Sex differences in plasmacytoid dendritic cell levels of IRF5 drive higher IFN-α..., Griesbeck [/bib_ref] , which has antiproliferative, differentiation-inducing, apoptotic, and antiangiogenic properties, and its clinical activity has been demonstrated in several cancers, including as postchemotherapy maintenance [bib_ref] Treating cancer with PEG intron: pharmacokinetic profile and dosing guidelines for an..., Bukowski [/bib_ref] [bib_ref] How much of a good thing? What duration for interferon alfa-2b adjuvant..., Tarhini [/bib_ref]. Zinc finger and BTB domain-containing 38 (ZBTB38) represents one member of the zinc finger (ZF) family of Methyl-CpG-binding proteins (MBPs) [bib_ref] Structural insights into methylated DNA recognition by the C-terminal zinc fingers of..., Hudson [/bib_ref]. Similar to the IRF5, in our experiment, its expression is related to cisplatin sensitivity. Previous study showed that ZBTB38 can enhance the response to DNMT inhibitor therapies as a target of DNA methyltransferase inhibitoras well as it can influence response of cancer cell lines to chemotherapy through involving in diverse epigenetic processes affecting DNA methylation [bib_ref] Association of expression of epigenetic molecular factors with DNA methylation and sensitivity..., Vural [/bib_ref]. However, The biological function of ZBTB38 remains also elusive [bib_ref] DNA methylation and chromatin: role(s) of methyl-CpG-binding protein ZBTB38, De Dieuleveult [/bib_ref]. In bladder cancer, ZBTB38 promotes migration and invasive growth [bib_ref] The role of ZBTB38 in promoting migration and invasive growth of bladder..., Jing [/bib_ref] , while in prostate cancer, depletion of ZBTB38 results in higher expression of ROS and elevated cell death after doxorubicin treatment [bib_ref] Molecular and clinical relevance of ZBTB38 expression levels in prostate cancer, De Dieuleveult [/bib_ref]. However, the specific mechanism among those 3 genes, with which cancer cells' sensitivity to cisplatin could be changed, still needs to be explored. miRNAs could modulate about 30% of gene expression through influencing mRNA translation [bib_ref] Mammalian microR-NAs predominantly act to decrease target mRNA levels, Guo [/bib_ref] , which play a key role in many biological processes, including tumor chemoresistance [bib_ref] Biological functions of microRNAs: a review, Huang [/bib_ref]. Evidence has shown that the expression of several miRNAs may relate to cisplatin resistance in malignant cells [bib_ref] Reduction of microRNA-184 by E6 oncoprotein confers cisplatin resistance in lung cancer..., Tung [/bib_ref] [bib_ref] MiR-1244 sensitizes the resistance of non-small cell lung cancer A549 cell to..., Li [/bib_ref]. In our study, miR-200c and miR-203 finally contributed to the cisplatin-resistance model. The former, which belongs to the miR-200 family, is reported that it could increase the sensitivity of cells to antitumor medications in a variety of cancers, including gastric [bib_ref] MicroRNA-200c regulates cisplatin resistance by targeting ZEB2 in human gastric cancer cells, Jiang [/bib_ref] [bib_ref] MicroRNA-200c regulates the sensitivity of chemotherapy of gastric cancer SGC7901/DDP cells by..., Chang [/bib_ref] , breast [bib_ref] MiR-200c suppresses TGF-β signaling and counteracts trastuzumab resistance and metastasis by targeting..., Bai [/bib_ref] , and nonsmall cell lung cancer [bib_ref] Identification of microRNA-based signatures for response and survival for non-small cell lung..., Berghmans [/bib_ref]. Similarly, miR-203 was differentially expressed in DDP-sensitive and -insensitive tumor cells. Previous study has demonstrated that miR-203 could bind to the 3′UTR of DKK1 and then regulate the characteristics of lung cancer cells [bib_ref] Overexpression of miR-203 increases the sensitivity of NSCLC A549/H460 cell lines to..., Cheng [/bib_ref]. Furthermore, it also affects cisplatin resistance of pancreatic cancer cells [bib_ref] MiR-203 regulates DJ-1 expression and affects proliferation, apoptosis and DDP resistance of..., Du [/bib_ref] , tongue squamous cancer [bib_ref] miR-203 inhibits cell proliferation and promotes cisplatin induced cell death in tongue..., Lin [/bib_ref]. In all, large numbers of surveys indicate that miRNAs actively affect the mechanism of cisplatin resistance. # Conclusions Somatic mutations, mRNA expressions, miRNA expressions, and metabolites differences were related to the resistance of cisplatin. The model we created and based on the expression of 3 genes, BATF3, IRF5, ZBTB38, could help in the prediction of the reaction and prognosis of cancer patients given platinum-based, especially cisplatin-including chemotherapies. [fig] Figure 1, Figure 2, Figure 3: Flow Somatic mutation difference between high and low IC50 groups of cisplatin; A refers to the low IC50 group; B for the high IC50 group; C for the compartments between 2 groups ranked by the rate of difference; D top 15 different mutations between 2 groups ranked by P-value Differential analysis from different levels between cisplatin sensitive and resistant group; A for the mRNA level; B for the miRNA level; C for the metabolism level; D for the methylation level; E-G Pathway analysis of the different coding genes; E for GO; F for KEGG; G for GSVA [/fig] [fig] Figure 4: cisplatin-sensitivity related model. A, B. Establishment of the LASSO model; C Coefficient display of logistics regression equation model of cisplatin-sensitivity related genes [/fig] [fig] Figure 5: K-M plot showing the differences of survival time between high and low expression of FOXA2, BATF3, SIX1, HOXA1, ZBTB38, IRF5 [/fig] [fig] Figure 6: Silencing BATF3 significantly increases sensitivity to cisplatin, while silencing IRF5, ZBTB38 reduces. A, B qRT-PCR and Western blot showing BATF3, IRF5, ZBTB38 knockdown 3 and 4 days after transfection with BATF3-targeting, IRF5-targeting and ZBTB38-targeting siRNAs with cytotoxicity curves of the lung adenocarcinoma cell line A549 and H358 transfected with nontargeting (ctrl) or BATF3-targeting, IRF5-targeting and ZBTB38-targeting siRNAs and treated for 48 h with cisplatin. C Immunohistochemistry for NACT sensitive and insensitive LUAD patients' [/fig] [fig] Figure 7: tSNE of tumor cells, BATF3, IRF5, ZBTB38 in NACT-NON-SEN and NACT-SEN groups in ESCC (A) and LUAD (B). Violin plots showing the difference of expression of the 3 genes between NACT sensitive and insensitive ESCC (C) or LUAD (D) patients for malignant cells [/fig] [table] Table 1: Coefficient display of logistics regression equation model for miRNA [/table]

Nuclear glycogenolysis modulates histone acetylation: a novel mechanism of epigenetic regulation in cancer Nuclear glycogenolysis modulates histone acetylation: a novel mechanism of epigenetic regulation in cancer Open Access Cancer [email protected] glycogenolysis modulates histone acetylation: a novel mechanism of epigenetic regulation in cancer Open Access Cancer Communications Dong Cancer Commun (2019) 39:71 RESEARCH HIGHLIGHT *Correspondence: Page 2 of 2 Dong Cancer Commun (2019) 39:71 which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Cancer cells, as compared to normal cells, have their own metabolic alterations [1] which are essential for their survival and proliferation as they have higher energy production [2], maintenance for redox potential[3], and anabolic pathway induction[4]. Thus, metabolic reprogramming has been considered as a potential therapeutic target of cancer. In recent years, several studies have shown evidences that metabolic reprogramming is regulated by the genetic events of cancer cells. However, as the metabolites of the cancer cells also simultaneously affect epigenetic events, these make cancer cells dependent on certain metabolic-related substances, such as lipids and glutamine[5].Glycogen, a multibranched polysaccharide of glucose, is the main storage form of glucose that can be broken down to yield glucose molecules when cells need energy. It is found in most normal tissues and accumulated in specific subcellular organelles. Several studies have reported that glycogen was elevated in multiple cancer cell lines, including lung [6], breast[7], and colorectal cancer[8]. Additionally, hypoxia, a key characteristic of tumor mass, can enhance carcinogenesis, suppress reactive oxygen species levels and inhibit senescence via inducing glycogenolysis in breast [7] and colorectal cancer[8]. These studies suggested that glycogen can serve as an important energy source in cancer cells to enable survival and proliferation under hypoxia. However, aside from being an energy source, the potential roles of glycogen in cancer are still unclear.In a recent study published in Cell Metabolism, entitled "Nuclear Glycogenolysis Modulates Histone Acetylation in Human Non-Small Cell Lung Cancers", Sun et al.[9]demonstrated that nuclear glycogen metabolism plays a critical role in providing substrates for histone acetylation in non-small cell lung cancer (NSCLC) and further explored how NSCLC cells can perturb this pathway to support proliferation under hypoxia. By using nuclei preparations and isotope tracers, the authors observed that nuclear glycogen was de novo synthesized in NSCLC cell lines to promote pyruvate pool formation for histone acetylation; thereby promoting the development and progression of NSCLC. Mechanism studies have shown that NSCLC cell lines can accumulate nuclear glycogen by down-regulating marlin, an E3 ubiquitin ligase that could enhance glycogenolysis, which were validated in animal xenograft models and 50 paired human NSCLC tissues and normal lung tissues. These data showed that nuclear glycogen is not only a polymer compound that serves as a form of energy storage but also a signaling molecule in NSCLC.One of the innovations of this study is that the authors used a novel combination method, combine isolation methods with highly sensitive tracking technologies, to detect the metabolism of specific organelles. In addition, this study demonstrates that nuclear glycogen of NSCLC cells provides a carbon source for histone acetylation, and E3 ubiquitin ligase and glycogen phosphorylase ubiquitination is involved in this metabolic process. In other words, the downregulation of glycogenolysis due to decreased malin and nuclear glycogen phosphorylase expression further leads to a lack of substrate for histone acetylation and ultimately results in an alteration of the epigenetic landscape seen in NSCLC. Based on these findings, the authors proposed a new concept that E3 ubiquitin ligase can serve as a signaling molecule by regulating glycogen metabolism in the nucleus of NSCLC cells.Mutations in metabolic enzymes result in the upregulation of oncometabolites, for instance, mutations in isocitrate dehydrogenase result in excess production of 2-hydroxyglutarate[10]. Nearly all of these oncometabolites have been reported to cause cancer malignancy by inhibiting DNA and/or histone demethylases[5]. Aberrant histone acetylation is a hallmark in multiple cancers, especially NSCLC. Vorinostat, a histone deacetylase inhibitor that can increase histone acetylation, has been demonstrated to have anti-cancer activity in NSCLC[11]. However, the mechanisms by which oncometabolites affect histone acetylation remains unclear. In normal cells, nuclear localization of some metabolic enzymes, such as adenosine triphosphate citrate lyase, pyruvate dehydrogenase, and acetyl-coenzyme A synthase, can supply histone acetylation[12][13][14]. Although these metabolic enzymes have been shown to promote histone acetylation by using pyruvate and acetate, the origin of their compartmentalized substrates still remains unknown. The authors have demonstrated that glycogen contributes to the formation of pyruvate and acetate pool in the nucleus. This study reveals an underlying molecular mechanism of an observation that has been in existence for more than 70 years; thereby redefining the role of nuclear glycogen in cancer biology and provided a new therapeutic target for NSCLC.Abbreviation NSCLC: non-small cell lung cancer.AcknowledgementsNot applicable. Cancer cells, as compared to normal cells, have their own metabolic alterationswhich are essential for their survival and proliferation as they have higher energy production, maintenance for redox potential, and anabolic pathway induction. Thus, metabolic reprogramming has been considered as a potential therapeutic target of cancer. In recent years, several studies have shown evidences that metabolic reprogramming is regulated by the genetic events of cancer cells. However, as the metabolites of the cancer cells also simultaneously affect epigenetic events, these make cancer cells dependent on certain metabolic-related substances, such as lipids and glutamine. Glycogen, a multibranched polysaccharide of glucose, is the main storage form of glucose that can be broken down to yield glucose molecules when cells need energy. It is found in most normal tissues and accumulated in specific subcellular organelles. Several studies have reported that glycogen was elevated in multiple cancer cell lines, including lung, breast, and colorectal cancer. Additionally, hypoxia, a key characteristic of tumor mass, can enhance carcinogenesis, suppress reactive oxygen species levels and inhibit senescence via inducing glycogenolysis in breastand colorectal cancer. These studies suggested that glycogen can serve as an important energy source in cancer cells to enable survival and proliferation under hypoxia. However, aside from being an energy source, the potential roles of glycogen in cancer are still unclear. In a recent study published in Cell Metabolism, entitled "Nuclear Glycogenolysis Modulates Histone Acetylation in Human Non-Small Cell Lung Cancers", Sun et al.demonstrated that nuclear glycogen metabolism plays a critical role in providing substrates for histone acetylation in non-small cell lung cancer (NSCLC) and further explored how NSCLC cells can perturb this pathway to support proliferation under hypoxia. By using nuclei preparations and isotope tracers, the authors observed that nuclear glycogen was de novo synthesized in NSCLC cell lines to promote pyruvate pool formation for histone acetylation; thereby promoting the development and progression of NSCLC. Mechanism studies have shown that NSCLC cell lines can accumulate nuclear glycogen by down-regulating marlin, an E3 ubiquitin ligase that could enhance glycogenolysis, which were validated in animal xenograft models and 50 paired human NSCLC tissues and normal lung tissues. These data showed that nuclear glycogen is not only a polymer compound that serves as a form of energy storage but also a signaling molecule in NSCLC. One of the innovations of this study is that the authors used a novel combination method, combine isolation methods with highly sensitive tracking technologies, to detect the metabolism of specific organelles. In addition, this study demonstrates that nuclear glycogen of NSCLC cells provides a carbon source for histone acetylation, and E3 ubiquitin ligase and glycogen phosphorylase ubiquitination is involved in this metabolic process. In other words, the downregulation of glycogenolysis due to decreased malin and nuclear glycogen phosphorylase expression further leads to a lack of substrate for histone acetylation and ultimately results in an alteration of the epigenetic landscape seen in NSCLC. Based on these findings, the authors proposed a new concept that E3 ubiquitin ligase can serve as a signaling molecule by regulating glycogen metabolism in the nucleus of NSCLC cells. Mutations in metabolic enzymes result in the upregulation of oncometabolites, for instance, mutations in isocitrate dehydrogenase result in excess production of 2-hydroxyglutarate. Nearly all of these oncometabolites have been reported to cause cancer malignancy by inhibiting DNA and/or histone demethylases. Aberrant histone acetylation is a hallmark in multiple cancers, especially NSCLC. Vorinostat, a histone deacetylase inhibitor that can increase histone acetylation, has been demonstrated to have anti-cancer activity in NSCLC. However, the mechanisms by which oncometabolites affect histone acetylation remains unclear. In normal cells, nuclear localization of some metabolic enzymes, such as adenosine triphosphate citrate lyase, pyruvate dehydrogenase, and acetyl-coenzyme A synthase, can supply histone acetylation. Although these metabolic enzymes have been shown to promote histone acetylation by using pyruvate and acetate, the origin of their compartmentalized substrates still remains unknown. The authors have demonstrated that glycogen contributes to the formation of pyruvate and acetate pool in the nucleus. This study reveals an underlying molecular mechanism of an observation that has been in existence for more than 70 years; thereby redefining the role of nuclear glycogen in cancer biology and provided a new therapeutic target for NSCLC. Abbreviation NSCLC: non-small cell lung cancer. AcknowledgementsNot applicable.Authors' contributionsThe author read and approved the final manuscript.FundingThis project is supported by Project of Tianjin Municipal Education Commission (No. 20140118).Availability of data and materialsNot applicable.Ethics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe author declares no competing interests.Received: 30 October 2019 Accepted: 1 November 2019

Seroconversion after anti‐SARS‐CoV‐2 mRNA vaccinations among moderate‐to‐severe psoriatic patients receiving systemic biologicals—Prospective observational cohort study It is unclear whether biological antipsoriatic therapies affect seroconversion after messenger ribonucleic acid (mRNA)-based antisevere acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) vaccinations. To assess antibody formation and the incidence of side effects after anti-SARS-CoV-2 mRNA vaccinations in psoriatic patients receiving different biologicals compared to healthy controls. 102 moderateto-severe psoriatic patients (56.2 [±13.5] years) and 55 age-matched healthy (56.4 ± 13.6 years) volunteers were included in our study. Ten to 21 days after the administration of the second dosage of BNT162b2 or mRNA-1273 vaccine, antibody levels specific to the SARS-CoV-2 spike (S) protein receptor binding domain were monitored. The incidence of postvaccination side effects was recorded and compared to real-life data in the literature. Of the 102 patients, 57 (55.88%) received tumor necrosis factor (TNF), 28 (27.45%) received interleukin (IL)-12/23, 16 (15.68%) received IL-17, and 1 (0.99%) received IL-23 inhibitors. No significant differences in the median serum level of anti-SARS-CoV-2S antibody were observed between the study population and the control group (median IQR range: 1681.0 U/mL (600.0-4844.0) versus 1984.0 U/mL (1000.0-3136.0; p = 0.82). The most frequent side effects of the mRNA vaccines within 7 days after the administration of both dosages were arm painon the side of injection (23.53% and 23.53%), fatigue (9.80% and 13.72%), headache (4.9% and 5.88%), and chills or shivering (4.9% and 8.82%). Detectable antibodies against SARS-CoV-2S protein appear 10-21 days after the administration of the second dosage of BNT162b2 or mRNA-1273 vaccines in moderate-to-severe psoriatic patients receiving biologicals, similar to those of healthy controls. , and it has steadily progressed into a global pandemic. [bib_ref] History is repeating itself: probable zoonotic spillover as the cause of the..., Rodriguez-Morales [/bib_ref] A large amount of data is already available about comorbidities, which lead to a poor outcome of COVID-19. [bib_ref] Epidemiology, clinical course, and outcomes of critically ill adults with COVID-19 in..., Cummings [/bib_ref] [bib_ref] Epidemiology of COVID-19: a systematic review and meta-analysis of clinical characteristics, risk..., Li [/bib_ref] [bib_ref] Epidemiology and outcomes of novel coronavirus 2019 in patients with immunemediated inflammatory..., Gianfrancesco [/bib_ref] Compared to the general population, psoriasis does not increase the risk of SARS-CoV-2 infection. [bib_ref] Covid-19 in immune-mediated inflammatory diseases -case series from New York, Haberman [/bib_ref] [bib_ref] Psoriasis, biological drugs and coronavirus disease 2019: real life experience of two..., Vispi [/bib_ref] However, psoriasis is often accompanied by several other comorbidities, such as metabolic syndrome and cardiovascular diseases, which on their own result in a poor COVID-19 outcome in the general population. [bib_ref] Clinical determinants for fatality of 44,672 patients with COVID-19, Deng [/bib_ref] [bib_ref] The effect of methotrexate and targeted immunosuppression on humoral and cellular immune..., Mahil [/bib_ref] In this study, the authors' aim was to investigate anti-SARS-CoV-2S antibody levels after the administration of the second dose of the anti-Budapest, Hungary (approval number: IV/861-1/2021/EKU, date of approval: January 27, 2021) and meets all requirements of the Declaration of Helsinki. Patients provided written informed consent to participate and donate blood for the purpose of our study. ## | study design This multicenter study investigated a prospective cohort of immuno- Patients were excluded if they had a polymerase chain reaction (PCR) proven current or previous SARS-CoV-2 infection, previous severe vaccination reaction (anaphylaxis), known primary immunodeficiency that affects adaptive immunity, status of splenectomy or functional asplenia, solid organ transplantation within 3 months, treatment targeting B-cell clones (anti-CD20), neutropenia (neutrophil granulocyte < 0.5 G/L), lymphopenia (lymphocyte count < 0.5 G/L), pregnancy or breast-feeding, or were planning to conceive a child within 2 months. Patients were also excluded if any of the following conditions were present: psoriatic disease for less than 6 months, induction period of the applied antipsoriatic systemic biological therapy, concomitant MTX therapy, or younger than 18 years. vaccination, 5 mL of blood samples were collected. Side effects were registered after both vaccinations. We examined the effects of these two vaccines together because the mechanism and efficacy are approximately the same. [bib_ref] National Psoriasis Foundation COVID-19 task force guidance for management of psoriatic disease..., Gelfand [/bib_ref] , [bib_ref] Advances in gene-based vaccine platforms to address the COVID-19 pandemic, Pushparajah [/bib_ref] We did not stratify the patients according to the type of mRNA vaccinations. Control group consisted of 55 volunteers who were, healthy agematched health care workers (45 women, 10 men) were enrolled in the study (age: 56.4 ± 13.6 years). The exclusion criteria were an autoimmune disease or positive SARS-CoV-2 antibody test results with the Roche test before vaccination. Specific antibody tests (Roche anti-SARS-CoV-2 S immunoassay) were used to examine the presence of antibodies 10-14 days after the administration of the second dose of mRNA vaccine. The incidence rates of side effects after the administration of both dosages of mRNA vaccines are shown in . ## | laboratory analyses ## | evaluation of data ## | laboratory findings The results of the laboratory examinations are shown in and . [table] 1: Characteristics of moderate-to-severe psoriatic patients receiving different biological therapies [/table]

Spontaneous Neural Dynamics and Multi-scale Network Organization Spontaneous neural activity has historically been viewed as task-irrelevant noise that should be controlled for via experimental design, and removed through data analysis. However, electrophysiology and functional MRI studies of spontaneous activity patterns, which have greatly increased in number over the past decade, have revealed a close correspondence between these intrinsic patterns and the structural network architecture of functional brain circuits. In particular, by analyzing the large-scale covariation of spontaneous hemodynamics, researchers are able to reliably identify functional networks in the human brain. Subsequent work has sought to identify the corresponding neural signatures via electrophysiological measurements, as this would elucidate the neural origin of spontaneous hemodynamics and would reveal the temporal dynamics of these processes across slower and faster timescales. Here we survey common approaches to quantifying spontaneous neural activity, reviewing their empirical success, and their correspondence with the findings of neuroimaging. We emphasize invasive electrophysiological measurements, which are amenable to amplitude-and phasebased analyses, and which can report variations in connectivity with high spatiotemporal precision. After summarizing key findings from the human brain, we survey work in animal models that display similar multi-scale properties. We highlight that, across many spatiotemporal scales, the covariance structure of spontaneous neural activity reflects structural properties of neural networks and dynamically tracks their functional repertoire.SPONTANEOUS BRAIN ACTIVITYAs an organ in perpetual action, the human brain consumes 20% of the body's metabolic budget, despite constituting only 2% of body weight(Raichle, 2010). This metabolic consumption supports both spontaneous processes, which occur independent of the immediate environment, as well as processes that are coupled to the external world. In systems neuroscience, the spontaneous component of brain activity has often been viewed as a kind of noise: an uninformative nuisance variable that should be controlled for in experimental design and removed during data analysis. However, it is clear that the anatomical structure and physiological processes of neurons and surrounding tissue impose constraints on the spatial and temporal properties of both Spontaneous neural activity has historically been viewed as task-irrelevant noise that should be controlled for via experimental design, and removed through data analysis. However, electrophysiology and functional MRI studies of spontaneous activity patterns, which have greatly increased in number over the past decade, have revealed a close correspondence between these intrinsic patterns and the structural network architecture of functional brain circuits. In particular, by analyzing the large-scale covariation of spontaneous hemodynamics, researchers are able to reliably identify functional networks in the human brain. Subsequent work has sought to identify the corresponding neural signatures via electrophysiological measurements, as this would elucidate the neural origin of spontaneous hemodynamics and would reveal the temporal dynamics of these processes across slower and faster timescales. Here we survey common approaches to quantifying spontaneous neural activity, reviewing their empirical success, and their correspondence with the findings of neuroimaging. We emphasize invasive electrophysiological measurements, which are amenable to amplitude-and phasebased analyses, and which can report variations in connectivity with high spatiotemporal precision. After summarizing key findings from the human brain, we survey work in animal models that display similar multi-scale properties. We highlight that, across many spatiotemporal scales, the covariance structure of spontaneous neural activity reflects structural properties of neural networks and dynamically tracks their functional repertoire. Keywords: resting-state fMRI, electrocorticography (ECoG), brain networks, connectivity, neural dynamics ## Spontaneous brain activity As an organ in perpetual action, the human brain consumes 20% of the body's metabolic budget, despite constituting only 2% of body weight [bib_ref] Two views of brain function, Raichle [/bib_ref]. This metabolic consumption supports both spontaneous processes, which occur independent of the immediate environment, as well as processes that are coupled to the external world. In systems neuroscience, the spontaneous component of brain activity has often been viewed as a kind of noise: an uninformative nuisance variable that should be controlled for in experimental design and removed during data analysis. However, it is clear that the anatomical structure and physiological processes of neurons and surrounding tissue impose constraints on the spatial and temporal properties of both spontaneous and task-related neural activity. Thus, spontaneous activity patterns should reflect these spatiotemporal constraints. A wealth of empirical data across different levels of investigation supports the notion that spontaneous neural activity is functionally relevant. Most recently, studies of the organization of spontaneous hemodynamic activity in the human brain using functional magnetic resonance imaging (fMRI) have provided reliable maps of covariation in cortical and subcortical activity [bib_ref] Functional network organization of the human brain, Power [/bib_ref] [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref]. This approach can extract and parcellate sets of covarying regions ("functional networks") that match the sets of regions co-active during task conditions. For this reason, and because the covariance analyses make novel predictions of previously underappreciated functional divisions, there is now widespread interest in the spatial organization and temporal dynamics of spontaneous brain activity [bib_ref] Dynamic functional connectivity: promise, issues, and interpretations, Hutchison [/bib_ref] [bib_ref] Studying brain organization via spontaneous fMRI signal, Power [/bib_ref]. In this Review, we outline existing evidence that brain networks identified via resting-state fMRI (rsfMRI) have clear electrophysiological correlates in the human brain. We highlight the most popular techniques used to study correlated patterns of spontaneous neuronal activity in the resting-state, and examine the contribution of these techniques to understanding the origin and implications of spontaneous cortical activity. Next we discuss how similar conceptual and analytical methods have previously been applied to neuronal and population scale electrophysiology. We highlight similar observations across studies of spontaneous connectivity (at the regional/macro scale) and studies of spontaneous noise correlations (in activity at the cellular/micro and circuit/meso-scale). Despite several orders of magnitude of difference in spatial scale, recent empirical work has highlighted that spontaneous brain activity can capture important structural and functional properties of local and global neural systems, consistent with the view that the statistical properties of spontaneous activity reflect anatomical and physiological constraints of organizational principles within the central nervous system. # Correlated spontaneous hemodynamics Hemodynamic activity in the mammalian brain displays a sensitive and complex relationship to local electrophysiological activity [bib_ref] Intracortical recordings and fMRI: an attempt to study operational modules and networks..., Logothetis [/bib_ref]. Utilizing the compensatory delivery of oxygenated hemoglobin to sites of elevated neural activity, blood oxygenation level dependent (BOLD) fMRI provides an indirect and non-invasive whole brain measurement of large-scale neural activity [bib_ref] Interpreting the BOLD signal, Logothetis [/bib_ref]. As hemodynamic responses are often consequent to local neural events, and modulated by cardio-vascular physiology, BOLD fMRI activity is a delayed, and slow time varying (<1 Hz) signal. Despite these limiting properties, spontaneous BOLD fMRI displays ongoing large amplitude signal variations, even when subjects are not performing any explicit task (e.g., awake, with eyes closed or fixating). Indeed, the spontaneous fluctuations in BOLD activity can be larger in magnitude than sensory-evoked responses [bib_ref] Two views of brain function, Raichle [/bib_ref]. Beginning with observations by [bib_ref] Functional connectivity in the motor cortex of resting human brain using echo-planar..., Biswal [/bib_ref] , empirical studies of spontaneous BOLD fMRI have revealed spatial covariance across cortical and subcortical structures that often conforms to previously established functional brain networks. For example, rsfMRI signal covariance can be used to parcellate sensorimotor systems [bib_ref] Specific somatotopic organization of functional connections of the primary motor network during..., Van Den [/bib_ref] [bib_ref] Dorsal anterior cingulate cortex modulates supplementary motor area in coordinated unimanual motor..., Asemi [/bib_ref] , higher order associative fronto-parietal networks [bib_ref] Coherent spontaneous activity identifies a hippocampal-parietal memory network, Vincent [/bib_ref] [bib_ref] Evidence for a frontoparietal control system revealed by intrinsic functional connectivity, Vincent [/bib_ref] , thalamic nuclei [bib_ref] Intrinsic functional relations between human cerebral cortex and thalamus, Zhang [/bib_ref] [bib_ref] Functional connectivity-based parcellation of the thalamus: an unsupervised clustering method and its..., Fan [/bib_ref] , and cerebellar cortex [bib_ref] The organization of the human cerebellum estimated by intrinsic functional connectivity, Buckner [/bib_ref]. In particular, studies quantifying correlational structures in spontaneous BOLD signals during non-task "resting-states" have grown exponentially over the past 20 years, resulting in detailed parcellations of functionally separated cortical networks in the human brain (e.g., [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref] [bib_ref] Studying brain organization via spontaneous fMRI signal, Power [/bib_ref]. A key factor in the continued and expanding interest in studying spontaneous fMRI signals has been the striking correspondence between resting-state functional networks and previously established maps of functional co-activation accumulated by cognitive neuroimaging (e.g., [bib_ref] Correspondence of the brain's functional architecture during activation and rest, Smith [/bib_ref] [bib_ref] Intrinsic and task-evoked network architectures of the human brain, Cole [/bib_ref]. This correspondence opens the exciting possibility that correlational structures in spontaneous fMRI data provide an economical and non-invasive assay of large-scale functional network organization . As noted above, a key biological determinate/constraint of the structure of spontaneous activity are the ontogenetic factors defining anatomical structure in the human brain [bib_ref] Operational principles of neurocognitive networks, Bressler [/bib_ref]. However, rsfMRI connectivity displays interesting departures from anatomically prescribed organization [bib_ref] Predicting human resting-state functional connectivity from structural connectivity, Honey [/bib_ref] , as some regions, linked through poly-synaptic pathways rather than direct connections, display strong spontaneous correlation (e.g., . It is in this regard that rsfMRI, and spontaneous neuronal activity in general, reflects both constraints imposed by anatomy, and constraints imposed by the context and history of neural events that sculpt functional networks. As a putative assay of functional brain network organization, and as a non-invasive technique relying on basic physiological properties of the nervous system, rsfMRI can also be employed for comparative studies across mammalian species [bib_ref] Monkey in the middle: why non-human primates are needed to bridge the..., Hutchison [/bib_ref] , across clinical populations , and within subjects to assess the influence of learning [bib_ref] Learning sculpts the spontaneous activity of the resting human brain, Lewis [/bib_ref] [bib_ref] Visual learning induces changes in resting-state fMRI multivariate pattern of information, Guidotti [/bib_ref]. However, rsfMRI investigations were met with initial skepticism [bib_ref] Unrest at rest: default activity and spontaneous network correlations, Buckner [/bib_ref] [bib_ref] Does the brain have a baseline? Why we should be resisting a..., Morcom [/bib_ref] due to technological challenges [bib_ref] Separating respiratory-variation-related fluctuations from neuronalactivity-related fluctuations in fMRI, Birn [/bib_ref] [bib_ref] Spurious but systematic correlations in functional connectivity MRI networks arise from subject..., Power [/bib_ref] [bib_ref] The influence of head motion on intrinsic functional connectivity MRI, Van Dijk [/bib_ref] , whose resolution defines important milestones of the field . Of particular note is that several researchers have highlighted the uncertain interpretation of rsfMRI because of the unstructured nature of resting-state cognition [bib_ref] Does the brain have a baseline? Why we should be resisting a..., Morcom [/bib_ref] , as well as the complex relationship between BOLD signal fluctuations and ongoing electrophysiological activity [bib_ref] Ongoing physiological processes in the cerebral cortex, Leopold [/bib_ref]. Below, we focus on the literature assessing the electrophysiological correlates of rsfMRI in human and non-human primates. We then highlight how spontaneous electrophysiological data from the micro-and meso-scopic (neurons and circuits) levels display similar organizational principles found on the macroscopic (whole brain) level. ## Electrophysiology of correlated spontaneous hemodynamics Human and non-human primate rsfMRI has suggested that networks of cortical and subcortical regions can be reliably identified by examining the covariance structure of spontaneous hemodynamics (e.g., [bib_ref] Functional network organization of the human brain, Power [/bib_ref] [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref] [bib_ref] Monkey in the middle: why non-human primates are needed to bridge the..., Hutchison [/bib_ref] [bib_ref] Studying brain organization via spontaneous fMRI signal, Power [/bib_ref]. Prior to large-scale efforts to map human brain connectivity with rsfMRI [bib_ref] The WU-Minn Human Connectome Project: an overview, Van Essen [/bib_ref] , a number of criticisms were raised regarding the neural authenticity of slow (<1 Hz) varying hemodynamics [bib_ref] A brief history of the resting state: the Washington University perspective, Snyder [/bib_ref]. Identifying the electrophysiological correlates of this hemodynamic activity is therefore important; both for providing a neuronal basis to rsfMRI-defined networks as well as for elucidating mechanisms that may drive rsfMRI dynamics. As discussed below, although experimentally challenging, invasive techniques provide rich spatial, temporal, and spectral information for exploring rsfMRI correlates (for reviews of non-invasive findings see [bib_ref] Intrinsic coupling modes: multiscale interactions in ongoing brain activity, Engel [/bib_ref] [bib_ref] The contribution of electrophysiology to functional connectivity mapping, Schölvinck [/bib_ref] [bib_ref] The relationship between MEG and fMRI, Hall [/bib_ref]. We begin by outlining the most common analytical techniques for quantifying inter-regional coupling in spontaneous electrophysiological data. ## Approaches to measuring coupling in spontaneous brain activity A standard approach for characterizing the spatial organization of spontaneous neural activity is to compute Pearson's correlation (r) between time series of activity, i.e., [formula] r = 1 n n t=1 (X t − X t ) · (Y t − Y t ) 1 n n t=1 (X t − X t ) 2 · 1 n n t=1 (Y t − Y t ) 2 [/formula] where X t and Y t are two times series of interest, with X t and Y t being their respective means. Thus, r measures the covariance between two signals normalized by the individual variance of each signal, a ratio that is bounded to the range [−1, 1]. Algebraic reformulation also depicts r as the mean of the dot product of standardized scores (z-score) for X and Y (i.e., the dot product moment correlation coefficient; (Lee [bib_ref] Thirteen ways to look at the correlation coefficient, Rodgers [/bib_ref]. As a linear parametric statistic, r assumes X and Y to be continuous variables that are normally distributed and that, if related, share a linear association. In addition, X and Y should not contain univariate or bivariate outliers, and display homoscedasticity (i.e., homogeneous variance of leastsquares residuals; [bib_ref] Robust correlation analyses: false positive and power validation using a new open..., Pernet [/bib_ref]. For rsfMRI data, correlated variables are typically the preprocessed fMRI BOLD time series from regions of interest such as spatially separated voxels or voxel-clusters. For electrophysiological data, which contains greater temporal resolution, first and second order spectral features, including amplitude and phase, are commonly studied. For electrophysiological data, correlational analyses focus on either the raw, broadband time series, band limited time series, or the amplitude of band-limited time series. Typically, correlation of broadband time series is not desirable as this signal conflates, non-uniformly, the spectral content of electrophysiological signals (i.e., due to their high power relative to higher frequencies, the lowest frequencies dominate the signal). A more common approach is to study correlations within and across band limited frequency ranges. Studies of band limited correlations typically focus on the amplitude or power within the selected frequency band. While a number of different time-frequency approaches can be employed toward this aim [bib_ref] Intrinsic coupling modes: multiscale interactions in ongoing brain activity, Engel [/bib_ref] , the procedure adopted by numerous studies seeks to isolate a frequency range of interest through filtering or convolution, and to extract the instantaneous (i.e., sample-wise) time-varying amplitude of the band limited signal. For example, an experimenter might seek to study the correlation between two regions in the alpha band range between 7 and10 Hz. First, the spontaneous raw time series of interest is band pass filtered between 7 and 10 Hz, using an appropriately parameterized filter. Next, one can obtain the instantaneous amplitude signal of this band-limited data by applying a Hilbert transform to the filtered time series to compute the analytic signal [formula] ζ t = X t + iH(X t ). [/formula] The alpha band analytic signal (ζ t ) is a complex valued time series (also obtainable via wavelet convolution [bib_ref] Fourier-, Hilbert-and wavelet-based signal analysis: are they really different approaches?, Bruns [/bib_ref] , where the real component (X t ) is the original band passed signal, and the imaginary component (iH(X t ) is the Hilbert transform of (X t ), which corresponds to a 90 - rotation of (X t ). It is important to note the time-frequency uncertainty produced by time series filtering/convolution, which make instantaneous time series only "estimates" of the band limited signal. Basic trigonometric functions can then be applied to the analytic signal to identify the instantaneous amplitude and phase of the band passed signal. To obtain the instantaneous amplitude (a t ), also termed the signal envelope, the absolute value of the complex analytic signal is taken at each time point. For complex numbers, the absolute value is the length of the vector between the origin and the coordinate of the real and imaginary values in the complex plane (following Pythagoras' theorem, see [fig_ref] FIGURE 1 |: Quantifying band limited amplitude for correlation analysis of spontaneous data [/fig_ref] [formula] a t = X 2 t + H(X t ) 2 . [/formula] Instantaneous amplitude a t , in this example, reflects the samplewise estimate of alpha band amplitude, which can be squared to obtain signal power. The resultant time series (i.e., the time-varying amplitude or power from two separate recording locations) can then be used as the signals to be correlated. Importantly, the amplitude time series constitutes a signal with its own spectral properties , which may contain both slow and fast dynamics [bib_ref] Very slow activity fluctuations in monkey visual cortex: implications for functional brain..., Leopold [/bib_ref] [bib_ref] Slow cortical dynamics and the accumulation of information over long timescales, Honey [/bib_ref]. This observation is important when considering the differences in time scale between hemodynamic (∼0.01-1 Hz) and electrophysiological (∼0.01-300+ Hz) data, and the endeavor to capture electrical correlates of hemodynamic activity. As detailed below, one approach is to extract the Other time frequency methods (filter-Hilbert approach shown here) can be employed, such as Wavelet convolution to achieve comparable results [bib_ref] Fourier-, Hilbert-and wavelet-based signal analysis: are they really different approaches?, Bruns [/bib_ref]. amplitude of a higher frequency band-limited signal across time, and to then measure slow-varying changes in this amplitude time series. The resultant time scale of slow varying changes (<1 Hz) in higher frequency neuronal activity (>1 Hz) matches that of the fMRI BOLD response [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] [bib_ref] Quasiperiodic fluctuations in default mode network electrophysiology, Ko [/bib_ref] [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref] , thus bridging hemodynamic and electrophysiological data . Another approach is to use the low-frequency raw-filtered electrophysiological signal [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref]. In addition to a priori seed based analysis (i.e., correlation between selected regions/electrodes), more data driven approaches such as independent components analysis (ICA) are FIGURE 2 | Extracting slow time scale variability of rapid electrophysiological dynamics. (A) BOLD fMRI activity is a slow time varying signal, due to its hemodynamic basis. Plot shows an example time course of BOLD fMRI activity (left) and the power spectrum of this signal (right). Together these plots highlight the low frequency (<1 Hz) content of BOLD fMRI. (B) In contrast, electrophysiological activity, such as ECoG, has a wide spectral content. However, standard ECoG recordings apply a high-pass filter limiting the study of ultra slow time scales (but see [bib_ref] Infra-slow fluctuations in electrophysiological recordings, blood-oxygenation-level-dependent signals, and psychophysical time series, Palva [/bib_ref]. Plots show a raw ECoG time series (left), with a band pass range 0.5-300 Hz, and its power spectrum (right). The power spectrum shows a lack of power at lower frequencies (<1 Hz), owing to the recording filters, and a progressive decrease in power for higher frequencies, as commonly observed. (C) To study frequency specific activity patterns and inter-regional correlations, raw time series are filtered to isolate the frequency range of interest. Plots show an alpha range filtered time series (left), with a band pass of 7-10 Hz, and its power spectrum (right). The isolated alpha band activity is a rapid time varying signal, however the amplitude of alpha activity shows a slower rate of change. (D) Plots show the amplitude (red, left) of the alpha band time series from (C) and its power spectrum (right). Because the alpha band amplitude time course is not subject to the filtering of the recorded raw signal, it can contain lower frequency spectral content. (E) Typically, BOLD fMRI activity >1 Hz is excluded because it contains a number of non-hemodynamic artifacts . To more closely align the time scales of hemodynamic and electrophysiological activity, the alpha band amplitude signal can be low pass filtered <1 Hz, to obtain a time series with similar spectral content as BOLD fMRI. Plots show the time course of alpha band amplitude (red) and its low pass filtered form (black; left). The power spectrum (right) of the slow time varying alpha band amplitude shows similar spectral content as the BOLD signal. As reviewed, this approach of focusing on slow time scale modulations of higher frequency activity provides a successful means of comparing hemodynamic and electrophysiological activity. Importantly, this approach can be applied to any electrophysiological frequency range of interest, and allows comparison across frequencies [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref]. All power spectra are calculated from extended time series from which example epochs are shown, and are normalized to the spectral maxima, with frequency shown on a log scale. commonly applied to spontaneous neural data [bib_ref] Advances and pitfalls in the analysis and interpretation of resting-state FMRI data, Cole [/bib_ref]. These unsupervised approaches seek to quantitatively identify unique spatio-temporal patterns of covariation, i.e., putative networks, without a priori selection of brain anatomy. ICA has been successfully applied to rsfMRI data [bib_ref] Advances and pitfalls in the analysis and interpretation of resting-state FMRI data, Cole [/bib_ref] , and to resting-state MEG/EEG data [bib_ref] The contribution of electrophysiology to functional connectivity mapping, Schölvinck [/bib_ref] [bib_ref] The relationship between MEG and fMRI, Hall [/bib_ref]. The variability and sparsity of spatial sampling in most invasive electrophysiological studies has limited the utility of ICA based methods, and they will not be discussed in detail here. As noted above, instantaneous phase can also be extracted and "correlated" between two regions of interest. From the analytic signal calculated above, phase (φ t ) is defined as the inverse tangent of the ratio between the imaginary and real values at each time point [formula] φ t = tan −1 (H(X t )/X t ) [/formula] Phase values are expressed in radians (or degrees), and capture the angular position (counter clockwise rotation), of the complex valued coordinate, assuming the values of [−π < θ ≤ π] (see [fig_ref] FIGURE 1 |: Quantifying band limited amplitude for correlation analysis of spontaneous data [/fig_ref]. By obtaining the instantaneous phase signal (7-10 Hz alpha band in this example) from two regions, one can test for the consistency of their relative phase position over time (i.e., their phase synchrony): [formula] Cφ = 1 n n t = 1 e i(△φ t ) [/formula] Here, the estimated phase consistency Cφ between two regions (e.g., regions X and Y) is the absolute circular mean value of phase vectors reflecting the angular phase difference between regions X and Y at each time point (△φ t = φX t − φY t ). As each phase vector has unit length, Cφ will range from [0 to 1], where 1 is perfect phase consistency. This estimate has been described as the phase locking value by [bib_ref] Measuring phase synchrony in brain signals, Lachaux [/bib_ref]. A number of similar approaches exist for estimating phase correlation, with a varying nomenclature used by different authors [bib_ref] Five methodological challenges in cognitive electrophysiology, Cohen [/bib_ref]. However, in most cases the temporal consistency of phase signals is estimated via circular statistics. Importantly, the estimation of phase consistency requires careful consideration of type I errors, which may arise due to low statistical power (e.g., limited sample size) or contaminated measurements (e.g., volume conduction). A number of methodological improvements have been suggested to address these concerns (e.g., [bib_ref] Phase lag index: assessment of functional connectivity from multi channel EEG and..., Stam [/bib_ref] [bib_ref] The pairwise phase consistency: a bias-free measure of rhythmic neuronal synchronization, Vinck [/bib_ref] [bib_ref] An improved index of phase-synchronization for electrophysiological data in the presence of..., Vinck [/bib_ref]. While the example here focuses on the phase consistency of an alpha band signal between two regions, coupling may also occur at different frequencies between regions (e.g., 10 and 20 Hz; [bib_ref] Detection of n: m phase locking from noisy data: application to magnetoencephalography, Tass [/bib_ref]. ## Summary of invasive findings Invasive cortical recordings in humans and non-human primates provide direct insight into spontaneous neural activity at the micro-, meso-, and macro-scopic scale. Using multisite recordings, correlations of electrophysiological activity between distant brain regions can be explored, and in many cases compared directly to rsfMRI data. Using the analytical techniques described in the previous section, several investigators have sought to identify robust correlates of spontaneous covariation in cortical activity. Obtaining simultaneous intracranial electrophysiology and fMRI data from the human brain poses a number of technical challenges such as interference and induction currents caused by MRI [bib_ref] Simultaneous intracranial EEG-fMRI in humans: protocol considerations and data quality, Carmichael [/bib_ref]. As a consequence, most human studies have instead focused on comparing non-simultaneous electrophysiological and fMRI measurements within subjects who are in a common task or state. For example, [bib_ref] Coupling between neuronal firing, field potentials, and FMRI in human auditory cortex, Mukamel [/bib_ref] recorded single units and local field potentials (LFPs) from auditory cortex of human subjects who were viewing a movie, and compared electrophysiological responses against BOLD signals recorded from auditory cortex of a separate group of subjects watching the same movie. The authors found that slow changes in spike rate and the amplitude of high-frequency (40-130 Hz) LFPs were both positively correlated with the BOLD signal, while the amplitude of low frequencies in the 5-15 Hz range were anticorrelated with the BOLD signal. These findings are broadly consistent with reports based on simultaneous BOLD-LFP recordings in animals [bib_ref] Neurophysiological investigation of the basis of the fMRI signal, Logothetis [/bib_ref] [bib_ref] Hemodynamic signals correlate tightly with synchronized gamma oscillations, Niessing [/bib_ref]. Knowledge of the electrophysiological correlates of BOLD activity at a single brain location does not necessarily reveal the electrophysiological processes underlying the spatially distributed functional networks observed using fMRI. As one of the first invasive electrophysiological investigations of the neural bases of rsfMRI networks in the human brain, [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] collected rsfMRI data in drug-resistant epilepsy patients undergoing pre-surgical evaluation using intracranial electrocorticogram (ECoG) electrodes (the main clinical setting for human intracranial studies). Resting-state fMRI data were collected either before the implantation of the ECoG electrodes or after the electrodes were removed and the epileptogenic zone resected. By co-registering the anatomical MRI with the computer-aided tomography (CT) scan, which documented the electrode locations, the authors spatially co-registered fMRI and ECoG data in each patient (an approach common to most studies reviewed here). In order to identify the neurophysiological correlates of rsfMRI networks, the authors decomposed the ECoG signal into different frequency bands, and evaluated both the raw signal and the band-limited amplitude at different frequencies. In addition, the authors compared the correlation of ECoG signals obtained during normal wakefulness, slow-wave sleep (SWS), and rapid-eye-movement (REM) sleep with rsfMRI signals. Looking across states of arousal and across frequency bands, [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] observed that the ECoG signal in the lowfrequency range-i.e., the delta (1-5 Hz) and sub-delta (<1 Hz) bands that constitute the so-called slow cortical potentials (SCPs), provided the best correlate of the networks defined by rsfMRI signals. This correspondence persisted regardless of whether the ECoG data were collected during wakefulness, SWS or REM sleep. The band-limited amplitude of the gamma frequency (50-100 Hz) range also demonstrated good correspondence with the fMRI resting-state signals, but this relationship was weaker than the SCPs, and it was abolished during SWS. These results revealed the SCP as a theretofore unknown correlate of the spontaneous fMRI signal. Based on these findings and previous research on the physiology of SCP, the authors proposed that the SCP may also be a good correlate of the fMRI signal more generally [bib_ref] The fMRI signal, slow cortical potential and consciousness, He [/bib_ref]. This prediction has been borne out by two recent animal studies employing simultaneous fMRI and DC-coupled LFP recordings [bib_ref] Optogenetic drive of neocortical pyramidal neurons generates fMRI signals that are correlated..., Kahn [/bib_ref]. The findings of [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] identified two temporally different correlates of rsfMRI, the SCP and the (much faster) gamma band. How might these different time scales of cortical dynamics be reconciled? Some insight into this question was provided by [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref]. In a similar investigation, [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] recorded spontaneous activity invasively from both hemispheres of the human cortex and studied long-range correlation between them. These authors focused their analyses on unit spiking activity and band-limited LFP power (BLP) changes, and, innovatively, the modulation of these signals at fast (>1 Hz), medium (0.1-1 Hz), and slow (<0.1 Hz) time scales. As noted above in , the amplitude/envelope of LFP BLP has unique spectral properties, which can be low-pass filtered to study slow fluctuations. In performing this analysis, the authors were able to consider how rapid neural dynamics, like spiking or gamma band activity, are modulated at slow time scales similar to fMRI BOLD dynamics. [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] reported that across their measures, the best correlate of long-range network activity was the slow (<0.1 Hz) modulation of spiking and gamma band (40-100 Hz) activity. For example, spatial selectivity and high correlation was found between bilateral auditory cortices for slow modulations of gamma band activity. These findings provide a link to the observations of [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] by indicating that gamma band correlations may be subject to slow modulations (like the SCP) that are shared between distant regions [bib_ref] The fMRI signal, slow cortical potential and consciousness, He [/bib_ref]. Importantly, these long-range slow modulation gamma band correlations differ from those alternatively proposed to be coherent between functional regions [bib_ref] A mechanism for cognitive dynamics: neuronal communication through neuronal coherence, Fries [/bib_ref] [bib_ref] Intrinsic coupling modes: multiscale interactions in ongoing brain activity, Engel [/bib_ref]. Similar to [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] , [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] observed that long range correlations in slow modulations of gamma BLP were preserved across waking and sleep states. However, one limitation in linking the findings of [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] to rsfMRI data is the lack of direct comparison to rsfMRI from the same subjects. More recent investigations have helped address this question by applying similar data analyses to [bib_ref] Coupling between neuronal firing rate, gamma LFP, and BOLD fMRI is related..., Nir [/bib_ref] in subjects for which both resting-state ECoG and fMRI data were available. [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] studied the spatial correlations observed with resting ECoG recordings from the lateral cortical surface (covering frontal, parietal, and temporal lobes) for fast (1-10 Hz) or slow (0.1-1 Hz) modulations of high gamma-range activity (50-150 Hz). These authors report that slow modulations of high gamma power provided the strongest inter-regional correlations, and the strongest similarity to rsfMRI interregional correlations assessed within the same subjects. [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] also report that this similarity extends to anti-correlations (discussed below) observed between regions, although the BOLD-ECoG correspondence was substantially weaker for interregional anti-correlations. While these findings provide support for the identification of overlapping resting-state functional networks with electrophysiology and fMRI techniques, it is important to also directly confirm that regions showing putative connectivity do share functional responses under specific task conditions. Addressing this question directly, [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref] recently quantified the electrophysiological correlates of spontaneous and task-based fMRI correlations in human parietal cortex. Specifically, these authors focused on simultaneous ECoG recordings from the medial and lateral parietal surface during an explicit task, as well as rest and sleep states. Across these states, [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref] quantified correlations across the parietal lobe of the amplitude of high-frequency activity (70-180 Hz). During task conditions that required autobiographical retrieval, the authors observed strong trial-wise correlations between the medial retrosplenial/posterior cingulate (RSC/PCC) region and the lateral angular gyrus (AG). These anatomical regions are key nodes of the default network, which displays reliable fMRI activation during episodic memory retrieval [bib_ref] Parietal lobe contributions to episodic memory retrieval, Wagner [/bib_ref] [bib_ref] Brain networks underlying episodic memory retrieval, Rugg [/bib_ref]. During resting and sleeping states, the authors further quantified the correlation between parietal regions in slow (<1 Hz) fluctuations of spontaneous high frequency amplitude (70-180 Hz), analogous to the approach employed by [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] and [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] , and found correlation patterns that largely resembled those observed during the retrieval task. Next, [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref] compared ECoG resting-state connectivity patterns (correlation matrices) with rsfMRI data acquired for each subject, showing significant positive correlations between both modalities. Together, these findings support the view that slow time scale electrophysiological activity can exhibit strong correspondence with rsfMRI connectivity patterns, and that these slow fluctuations include the modulation of local high-frequency dynamics. In addition, these data also support the observation that spontaneous activity captures intrinsic properties of functional networks across behavioral states (e.g., task, rest, and sleep; see also [bib_ref] Emergence of sensory patterns during sleep highlights differential dynamics of REM and..., Ramot [/bib_ref]. However, future work is required to better understand the cellular and circuitlevel mechanisms driving these inter-regional correlations. One avenue for investigation toward this aim is the use of single cell recordings in non-human primates combined with fMRI imaging. Cortical resting-state activity has been evaluated at the level of single neurons using simultaneous fMRI and intracortical neurophysiological recordings, typically in anesthetized macaque monkeys. In one study focused on the primary visual cortex (V1), macaques were either exposed to a uniform gray field or kept in complete darkness [bib_ref] Neuronal correlates of spontaneous fluctuations in fMRI signals in monkey visual cortex:..., Shmuel [/bib_ref]. Under these conditions, the authors found that slow fluctuations in the BOLD signal were correlated with local neuronal activity following a (∼6 s) time lag that roughly resembled the hemodynamic response function (i.e., the time it takes vascular tissue to respond to local changes in metabolic demand). Activity in the gamma frequency band (24-90 Hz) as well as the local population spiking showed high correlations with the BOLD response. When the authors correlated the time-varying amplitude of these measures of local neurophysiological activity with the fMRI time-courses of voxels across the entire visual cortex, they found widespread coactivation of visual cortical areas across both hemispheres. These results suggest that, at least for the visual system, rsfMRI-based networks might be linked to synchronization of slow fluctuations in spiking activity across anatomically distinct, but functionally related brain areas (see [bib_ref] How not to study spontaneous activity, Logothetis [/bib_ref] for caveats). Although previous work suggests spontaneous correlations are intrinsically preserved across different states of awareness [bib_ref] Robust long-range coordination of spontaneous neural activity in waking, sleep and anesthesia, Liu [/bib_ref] , the effect of anesthetic compounds likely influences the covariation structure of spontaneous dynamics. More recently, simultaneous rsfMRI and neurophysiological recordings have been performed in alert non-human primates resting in a dark environment [bib_ref] Neural basis of global resting-state fMRI activity, Schölvinck [/bib_ref]. The authors focused on widespread, positive correlations of fMRI signals that are frequently excluded from resting-state functional connectivity analyses to correct for motion artifacts. Strikingly, the authors observed that the gamma-range activity (40-80 Hz) measured at a single cortical site was well-correlated with this global rsfMRI signal [bib_ref] Neural basis of global resting-state fMRI activity, Schölvinck [/bib_ref]. More specifically, the neuronal signal recorded at a single cortical site accounted for up to 10% of the global BOLD signal variance, suggesting a neuronal origin for these brain-wide BOLD correlations at rest. Taken together, the findings from these two studies suggest that fMRI fluctuations measured during the resting-state are closely linked to neural activity. The observation that spontaneous fluctuations in gamma range activity correlates with rsfMRI signals in non-human primates is consistent with studies observing a tight coupling between local BOLD activation and gamma band activity recorded from the same brain area in humans. As discussed above, human ECoG studies have reported that inter-areal correlations in gamma power are linked to BOLD correlations between these areas [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] [bib_ref] Identifying functional networks using endogenous connectivity in gamma band electrocorticography, Ko [/bib_ref] [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref]. An important technical qualification is that few studies use the same frequency range to define gamma activity, or the same terminology to describe frequencies between 40 and 200 Hz (e.g., gamma, high-gamma, broadband). Importantly, despite these differences, growing evidence suggests that changes in high frequency activity >40 Hz recorded from the human cortex has a broadband spectral representation, and the different subsampling of this frequency range will approximately track the same temporal process [bib_ref] Broadband changes in the cortical surface potential track activation of functionally diverse..., Miller [/bib_ref]. Importantly, high-frequency spectral changes also track event-related cortical deactivations [bib_ref] Negative functional MRI response correlates with decreases in neuronal activity in monkey..., Shmuel [/bib_ref] [bib_ref] Silence is golden: transient neural deactivation in the prefrontal cortex during attentive..., Lachaux [/bib_ref] [bib_ref] A widely distributed spectral signature of task-negative electrocorticography responses revealed during a..., Ramot [/bib_ref] , particularly within cortical regions comprising the default network, in both humans [bib_ref] Exploring the electrophysiological correlates of the default-mode network with intracerebral EEG, Jerbi [/bib_ref] [bib_ref] Differential electrophysiological response during rest, self-referential, and non-self-referential tasks in human posteromedial..., Dastjerdi [/bib_ref] [bib_ref] Transient suppression of broadband gamma power in the defaultmode network is correlated..., Ossandón [/bib_ref] and non-human primates [bib_ref] Electrophysiological correlates of default-mode processing in macaque posterior cingulate cortex, Hayden [/bib_ref]. While debate still surrounds the appropriate biophysical interpretation and statistical treatment of this spectral range [bib_ref] Gamma or no gamma, that is the question, Brunet [/bib_ref] [bib_ref] A unifying principle underlying the extracellular field potential spectral responses in the..., Podvalny [/bib_ref] [bib_ref] Do gamma oscillations play a role in cerebral cortex?, Ray [/bib_ref] [bib_ref] Interpreting the electrophysiological power spectrum, Gao [/bib_ref] , growing evidence suggests there are distinct neural processes that generate spectrally isolated (oscillatory) gamma and spectrally broad (asynchronous) high-frequency components that appear in the >40 Hz range [bib_ref] The origin of extracellular fields and currents-EEG, ECoG, LFP and spikes, Buzsáki [/bib_ref] [bib_ref] High-frequency neural activity and human cognition: past, present and possible future of..., Lachaux [/bib_ref] [bib_ref] Do gamma oscillations play a role in cerebral cortex?, Ray [/bib_ref]. In addition to correlated changes in the "gamma" range, several studies reviewed above also showed significant, although more spatially diffuse, correlations in the power of lower frequency oscillations between brain areas. Electrodes on the cortical surface used for ECoG studies most likely reflect activity of neurons in the supra-granular cortical layers [bib_ref] How local is the local field potential?, Kajikawa [/bib_ref] [bib_ref] Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the..., Fukushima [/bib_ref]. In contrast, studies in non-human primates (e.g., [bib_ref] Local origin of field potentials in visual cortex, Katzner [/bib_ref] measure activity in superficial or deep cortical layers, depending on electrode depth. There is growing evidence that different cortical layers show different spectral profiles, for example, higher gamma power in superficial layers, and more prominent low frequency activity in the deep layers [bib_ref] Laminar differences in gamma and alpha coherence in the ventral stream, Buffalo [/bib_ref] [bib_ref] Laminar analysis of visually evoked activity in the primary visual cortex, Xing [/bib_ref] [bib_ref] Laminar dependence of neuronal correlations in visual cortex, Smith [/bib_ref] [bib_ref] Microcircuitry of agranular frontal cortex: testing the generality of the canonical cortical..., Godlove [/bib_ref] [bib_ref] Microcircuitry of agranular frontal cortex: contrasting laminar connectivity between occipital and frontal..., Ninomiya [/bib_ref] ; but see [bib_ref] An oscillatory hierarchy controlling neuronal excitability and stimulus processing in the auditory..., Lakatos [/bib_ref] [bib_ref] Entrainment of neuronal oscillations as a mechanism of attentional selection, Lakatos [/bib_ref]. Low frequency oscillations (e.g., in the theta and alpha range) are generated by certain neuronal populations in the deep layers of cortex (Lopes Da [bib_ref] The cortical source of the alpha rhythm, Lopes Da Silva [/bib_ref] [bib_ref] Neural Mechanisms Underlying Brain Waves -from Neural Membranes to Networks, Lopes Da Silva [/bib_ref] [bib_ref] Intrinsic oscillations of neocortex generated by layer 5 pyramidal neurons, Silva [/bib_ref] [bib_ref] Neuronal mechanisms of cortical alpha oscillations in awake-behaving macaques, Bollimunta [/bib_ref] [bib_ref] Neuronal mechanisms and attentional modulation of corticothalamic alpha oscillations, Bollimunta [/bib_ref] [bib_ref] Layer-specific network oscillation and spatiotemporal receptive field in the visual cortex, Sun [/bib_ref] as well as in the thalamus [bib_ref] Thalamic mechanisms of EEG alpha rhythms and their pathological implications, Hughes [/bib_ref] [bib_ref] Cellular dynamics of cholinergically induced alpha (8-13 Hz) rhythms in sensory thalamic..., Lörincz [/bib_ref] , which projects to a subset of cortical layers. Low-frequency contributions to the BOLD signal thus may be more difficult to detect in studies that do not independently sample form the different cortical layers. To measure how much low-frequency activity contributes to rsfMRI correlations, [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] investigated a higher-order thalamocortical network. To do so, [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] simultaneously recorded neuronal activity from four distributed sites in visual cortex (V4), inferior temporal cortex (TEO), posterior parietal cortex (LIP), and the thalamus (pulvinar) of macaque monkeys during the resting-state. The authors compared this electrophysiological network activity to correlations in rsfMRI BOLD signals acquired under the same conditions. Analyses of slow-varying changes in LFP BLP, as well as the phase of LFPs, showed that low frequency activity (<20 Hz) predicted resting-state BOLD correlations between all pairs of network sites. Specifically, on a fast time-scale, [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] showed the highest coherence between neural activity from distant sites to be at theta/alpha frequencies. Similar to the analyses described above for human ECoG data, the authors also filtered time series of theta/alpha power into timescales matching the slow fluctuations of the BOLD signal (0.01-0.1 Hz), and found that the highest correlations between these band limited power time series also occurred at theta/alpha frequencies. Noninvasive work in human subjects provides further support for a broad range of frequencies, in addition to gamma, whose interregional coupling is associated with the correlations observed in rsfMRI [bib_ref] Electroencephalographic signatures of attentional and cognitive default modes in spontaneous brain activity..., Laufs [/bib_ref] [bib_ref] Electrophysiological signatures of resting state networks in the human brain, Mantini [/bib_ref] [bib_ref] Temporal dynamics of spontaneous MEG activity in brain networks, De Pasquale [/bib_ref] [bib_ref] Investigating the electrophysiological basis of resting state networks using magnetoencephalography, Brookes [/bib_ref] [bib_ref] Frequency specific interactions of MEG resting state activity within and across brain..., Marzetti [/bib_ref] [bib_ref] Exploring mechanisms of spontaneous functional connectivity in MEG: how delayed network interactions..., Cabral [/bib_ref] [bib_ref] BOLD fMRI Correlation Reflects frequency-specific neuronal correlation, Hipp [/bib_ref]. The prominent role of low frequency oscillations can be reconciled with evidence of gamma contributions to BOLD signals by considering cross-frequency coupling mechanisms (i.e., modulation of higher frequency components by lower frequencies). [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] investigated this possibility by first calculating the phase coherence between the phase of low frequency (alpha-range 8-13 Hz) oscillations between recordings sites. Secondly, the authors calculated how the phase of this low frequency oscillation modulated local high frequency gamma activity within each recording site [bib_ref] Assessing transient cross-frequency coupling in EEG data, Cohen [/bib_ref]. This crossfrequency coupling analysis suggested that the phase of low frequency oscillations was coherent between sites, and that it locally modulated the amplitude of gamma oscillations within sites . This suggests that the long-range correlation of gamma power between brain areas may be driven by lower frequency (e.g., alpha) coherence between regions, which locally modulate ongoing gamma power. The findings reviewed above beg the question of how neurons in different brain areas synchronize. There are extensive and reciprocal anatomical connections between the thalamus and cortex, supporting the notion that thalamocortical interactions are important for generating synchronous oscillatory activity across the brain. In addition to the first-order sensory relay nuclei in the thalamus (e.g., lateral geniculate, ventral posterior nuclei), which receive input from cortical layer 6 only, there are also higher-order thalamic nuclei (e.g., pulvinar, mediodorsal nucleus), which constitute the major volume of the thalamus and receive input from cortical layers 5 and 6. The higher-order thalamus respectively projects to cortical layer 4 and more superficial layers, providing indirect pathways between cortical areas, which are well-positioned to influence functional connectivity across the cortex [bib_ref] The functional logic of cortico-pulvinar connections, Shipp [/bib_ref] [bib_ref] Synchrony in the interconnected circuitry of the thalamus and cerebral cortex, Jones [/bib_ref] [bib_ref] Intralaminar and medial thalamic influence on cortical synchrony, information transmission and cognition, Saalmann [/bib_ref]. As mentioned above, the thalamus generates low frequency oscillations, e.g., alpha [bib_ref] Synchronized oscillations at alpha and theta frequencies in the lateral geniculate nucleus, Hughes [/bib_ref] [bib_ref] Thalamic mechanisms of EEG alpha rhythms and their pathological implications, Hughes [/bib_ref] [bib_ref] Cellular dynamics of cholinergically induced alpha (8-13 Hz) rhythms in sensory thalamic..., Lörincz [/bib_ref] , and thalamic lesions affect low frequency oscillations in the cortex [bib_ref] Thalamic control of spontaneous alpha-rhythm and evoked responses, Ohmoto [/bib_ref]. Other studies have shown that the pulvinar regulates the degree of alpha-and low-beta synchrony between visual cortical areas based on behavioral demands . Consistent with the idea of a closely coupled thalamocortical system, human and monkey studies have reported robust resting-state networks incorporating the thalamus [bib_ref] Intrinsic functional relations between human cerebral cortex and thalamus, Zhang [/bib_ref] [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref]. This suggests that the thalamus may be a vital node for supporting resting-state networks. In addition to low frequency subcortically mediated synchrony, long-range coordination may also be achieved through cortico-cortical high-frequency synchrony [bib_ref] Rhythms for cognition: communication through coherence, Fries [/bib_ref] [bib_ref] Communication through coherence with inter-areal delays, Bastos [/bib_ref]. A number of investigations have specifically highlighted the role of gamma range (40-80 Hz) synchrony in dynamic functional brain networks (e.g., [bib_ref] Visual areas exert feedforward and feedback influences through distinct frequency channels, Bastos [/bib_ref] , which provides an alternative mechanism for coordinating intrinsic network patterns [bib_ref] Intrinsic coupling modes: multiscale interactions in ongoing brain activity, Engel [/bib_ref]. While extant data has chiefly focused on the role of gamma synchrony during task conditions, it is of future interest to consider the spontaneous organization of gamma synchrony patterns, and to see how these rapid long-range dynamics may relate to the slower time scales of modulation described above [bib_ref] Interareal oscillatory synchronization in top-down neocortical processing, Bressler [/bib_ref] [bib_ref] Long-Range attention networks: circuit motifs underlying endogenously controlled stimulus selection, Womelsdorf [/bib_ref]. ## Correlated spontaneous activity across different scales of brain organization Invasive and non-invasive electrophysiological recordings from both humans and non-human primates suggest a number of, possibly related, neuronal correlates of spontaneous hemodynamic activity observed with rsfMRI. While comprehensive electrocortical mapping is challenging, focused efforts on specific networks suggest a strong overlap between the macro-scale areal parcellation of functional cortical regions through covariance structures in spontaneous activity using either modality [bib_ref] Electrophysiological correlates of the brain's intrinsic large-scale functional architecture, He [/bib_ref] [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref]. Given the striking correspondence between spontaneous and task-based parcellations of large-scale functional brain networks, it is of interest to consider the extent to which spontaneous activity may also reveal mesoand possibly micro-scale organization within brain regions. For example, sensory cortices display robust long-range correlations, such as spontaneous correlations between primary auditory or visual areas [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref]. However, within these areas there is well-documented topological organization (i.e., retinotopy or tonotopy). Does spontaneous activity, when measured with sufficient spatial resolution, conform to this within-region topology? Recent electrophysiological investigations suggest that spontaneous cortical activity on the mesoscopic scale also captures local, within-region functional organization. A useful bridging example between inter-areal and intra-areal organization, is provided by [bib_ref] Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the..., Fukushima [/bib_ref]. [bib_ref] Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the..., Fukushima [/bib_ref] utilized micro-ECoG recordings from the plane of the superior temporal sulcus in macaque monkeys to study evoked and spontaneous electrocortical activity within tonotopically organized auditory cortex. First, [bib_ref] Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the..., Fukushima [/bib_ref] established the ability to identify tonotopic cortical organization using task-evoked responses. Importantly, the authors focused on changes in the high-gamma range (50-150 Hz), analogous to the studies reviewed above. Using these task-based tonotopic maps as functional templates; the authors studied the spatial organization of spontaneous activity, again focusing on changes in high-gamma amplitude. Following this approach, the authors found high similarity between the spatial configuration of spontaneous and task-based activity patterns, such that spontaneous data could be used to estimate the predominant tonotopic organizational structure within auditory cortex. Analogous to the human ECoG work reviewed above, the findings of [bib_ref] Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the..., Fukushima [/bib_ref] suggest a pivotal role for spontaneous high-frequency activity (high-gamma, 50-150 Hz). Importantly, these authors focused on the direct changes in high-frequency amplitude, rather than slower fluctuations of high-frequency amplitude/power [bib_ref] Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex, Nir [/bib_ref] [bib_ref] Electrophysiological low-frequency coherence and cross-frequency coupling contribute to BOLD connectivity, Wang [/bib_ref] [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] [bib_ref] Intrinsic and taskdependent coupling of neuronal population activity in human parietal cortex, Foster [/bib_ref]. This difference in time-scale likely influences efforts to study different spatial scales of covariation, as low-pass filtering of the high-frequency amplitudes removes more localized activity, and instead favors slower, more diffusely correlated modulation of distant regions. Under this view, there are likely heterogeneous time scales of modulation present within spontaneous activity patterns, which relate to the different spatial and temporal constraints of local and distal network organization. This "nesting" of temporal dynamics across spatial scales is an important area for future investigation, however, there is some evidence suggesting infraslow EEG activity appears to modulate higher frequency activity, and also correlate with rsfMRI networks [bib_ref] Very slow EEG fluctuations predict the dynamics of stimulus detection and oscillation..., Monto [/bib_ref] [bib_ref] Infra-slow fluctuations in electrophysiological recordings, blood-oxygenation-level-dependent signals, and psychophysical time series, Palva [/bib_ref] [bib_ref] Infra-slow EEG fluctuations are correlated with resting-state network dynamics in fMRI, Hiltunen [/bib_ref]. While both human ECoG and micro-ECoG measurements in macaques provide clear evidence for strong correspondence between local spontaneous and task-related patterns on the level of neuronal population activity, such correspondence has also been documented at the level of single neurons. In a series of seminal investigations, Arieli et al. reported a striking correspondence between the spatial organization of spontaneous and task-evoked activity by triggering optical imaging data of the primary visual cortex in the cat to the spiking activity of isolated neurons [bib_ref] Coherent spatiotemporal patterns of ongoing activity revealed by real-time optical imaging coupled..., Arieli [/bib_ref] [bib_ref] Dynamics of ongoing activity: explanation of the large variability in evoked cortical..., Arieli [/bib_ref] , Specifically, they found that "snapshots" of local cortical activation at the time of elicited spikes were nearly indistinguishable between spontaneous and task-related responses. This correspondence between ongoing spontaneous activity and evoked responses, has been extended and replicated in visual [bib_ref] Linking spontaneous activity of single cortical neurons and the underlying functional architecture, Tsodyks [/bib_ref] [bib_ref] Spontaneously emerging cortical representations of visual attributes, Kenet [/bib_ref] as well as in somatosensory cortices [bib_ref] The relationship of anatomical and functional connectivity to restingstate connectivity in primate..., Wang [/bib_ref]. These studies, combined, suggest that the influence of spontaneous activity on task-evoked responses is a critical factor for the variability of neural responses across repeated stimulus presentations (see discussion below). Interestingly, these studies show that the similarity between spontaneous activity patterns and task-evoked functional organization progressively increases over early development, indicating shared sensitivity to anatomical and functional sculpting of the underlying neural tissue [bib_ref] Small modulation of ongoing cortical dynamics by sensory input during natural vision, Fiser [/bib_ref] [bib_ref] Spontaneous cortical activity reveals hallmarks of an optimal internal model of the..., Berkes [/bib_ref]. Therefore, in the developed brain, spontaneous activity reflects basic organizational properties of local neural populations, and at the same time influences the response properties of that population. This influence exerted by spontaneous activity can be a non-trivial factor on the computational properties of neural populations. Indeed a large body of work has focused on studying the influence of "noise-correlations" on population coding [bib_ref] Neural correlations, population coding and computation, Averbeck [/bib_ref]. The fact that spontaneous and task-elicited dynamics exhibit common patterns of covariance would not be especially surprising if one were to consider both as being determined by a common underlying anatomical connectivity. However, it can be more useful to think of monosynaptic anatomy as shaping and constraining, but not determining, the covariation in population activity [bib_ref] Predicting human resting-state functional connectivity from structural connectivity, Honey [/bib_ref]. A clear example of this principle is provided in the visual cortex, where task-driven and spontaneous BOLD fluctuations are generally correlated among regions and subregions that are known to be anatomically connected [bib_ref] Widespread functional connectivity and fMRI fluctuations in human visual cortex in the..., Nir [/bib_ref] [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref]. At the same time, however, there are departures from a direct correspondence: inter-regional correlations are stronger among subpopulations that represent the same visual eccentricity, more so than sites that represent the same polar angle [bib_ref] Widespread correlation patterns of fMRI signal across visual cortex reflect eccentricity organization, Arcaro [/bib_ref]. For example, the functional connectivity between circuits in human V2 and V3 reflect much more than the presumed monosynaptic reciprocal connections between neurons with common receptive fields. Similarly, as noted by [bib_ref] Functional connectivity patterns of visual cortex reflect its anatomical organization, Genç [/bib_ref] , the fact that foveal and peripheral sites within V1 are correlated despite lacking monosynaptic connections, again argues against monosynaptic links as the only driver of covariance, even within brain areas. Thus, anatomy shapes covariance, but does not constrain it to immediate first order interactions, allowing local and global network covariance. Indeed, for slow hemodynamic signals such as BOLD fMRI, there is ample room for covariance to be shaped by multi-synaptic causal paths, by common input to causally unconnected sites (e.g., from subcortical modulators), and, finally, as we discuss elsewhere in this review, by multi-scale emergent dynamical processes in the primate brain. ## Neural noise correlations One striking property of neuronal responses is the degree of variability in spiking activity across repeated presentations of a sensory stimulus. This trial-by-trial response variability is shared across specific members of the neuronal population [bib_ref] Measuring and interpreting neuronal correlations, Cohen [/bib_ref]. The degree of shared variability (around the mean response) across trials between neurons is typically referred to as noise correlation [bib_ref] Neural correlations, population coding and computation, Averbeck [/bib_ref] [bib_ref] Measuring and interpreting neuronal correlations, Cohen [/bib_ref]. The somewhat pejorative use of the term noise to describe these correlations has an historical explanation, as inter-regional and inter-neuronal correlations were originally studied from the perspective of the information capacity of population codes. In the language of Shannon's theory of information, a high level of independent spiking across a population of neurons carries a high amount of information [bib_ref] Correlated neuronal discharge rate and its implications for psychophysical performance, Zohary [/bib_ref]. Conversely, as the level of noise correlation increases across a population, the informational content that could theoretically be decoded at the next stage of processing (which receives this population activity readout as sole input) is reduced as similarly firing neurons become redundant [bib_ref] Neural correlations, population coding and computation, Averbeck [/bib_ref]. Experimental and computational work has extended this simplistic view to a more nuanced account of how noise correlations relate to the functional repertoire of neuronal populations and their relative size [bib_ref] Correlations and brain states: from electrophysiology to functional imaging, Kohn [/bib_ref]. Importantly, it has become clear that the degree of noise correlations during experimental stimulation provides insight into the shared interactions and inputs between members of local populations [bib_ref] Spontaneous and driven cortical activity: implications for computation, Ringach [/bib_ref]. In addition, more recent evidence suggests that these properties are present outside of task performance [bib_ref] Diverse coupling of neurons to populations in sensory cortex, Okun [/bib_ref] , and show important sensitivities to the context of behavioral states [bib_ref] State dependence of noise correlations in macaque primary visual cortex, Ecker [/bib_ref] [bib_ref] Cortical state determines global variability and correlations in visual cortex, Schölvinck [/bib_ref]. Above, we reviewed work that identified topographical organization in patterns of spontaneous activity at the surface of sensory cortex. It is of great interest whether or not spontaneous activity covariation may inform functional organization of nonsensory, associative cortical areas. A key challenge here is the uncertainty that functional organization conforms to cortical topography. Consideration of noise correlations has shown to be useful in this regard. For example, [bib_ref] Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex, Kiani [/bib_ref] recently addressed these questions directly by studying the statistical grouping of neurons in frontal cortex of the macaque. [bib_ref] Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex, Kiani [/bib_ref] studied the correlation of spiking activity between isolated pairs of neurons recorded across a high-density electrode array (Utah array) implanted in the prearcuate gyrus. Using unsupervised data clustering methods, the authors found that neuronal spiking during a visual task was functionally clustered within the prearcuate gyrus. Functional clustering was defined by the similarity of spiking responses across units. There were approximately two clusters of neurons showing dissociable response properties, which were also spatially dissociated across the recording array over the prearcuate gyrus. Importantly, the authors observe this clustering structure across all periods of the task, collectively (i.e., entire task) and in isolation (i.e., different trial periods). Neuronal response similarity across different task periods supports the view that the correlations driving clustering were due to shared "noise correlations" between neurons. To quantitatively confirm this inference, [bib_ref] Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex, Kiani [/bib_ref] analyzed the data focused only on the residual responses across trials, by subtracting the mean normalized activity during the task. This approach follows analytical approaches outlined above, where response variability across trials is correlated between neurons to estimate their shared "noise." The authors emphasized the analytical similarities between their work and common methods employed for large-scale parcellation of functional regions using rsfMRI. Importantly, the findings of [bib_ref] Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex, Kiani [/bib_ref] suggest that spontaneous activity is informative for functional grouping at the level of single neurons within higher order associative cortices for which topographical organizational principles are challenging to elucidate. It is of importance to understand how these local observations can be linked to the large-scale parcellations/networks observed with fMRI and electrophysiology. However, one promising observation was that similar parcellation across the recording array could be achieved using low frequency activity [bib_ref] Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex, Kiani [/bib_ref]. ## Dynamic structure of correlated spontaneous activity As reviewed above, spontaneous neural activity at multiple scales of cortical organization contains spatial patterns of covariant activity that reflect functional and anatomic constraints. Given this correspondence, and the reliability of topological cortical organization, it is worth considering whether the correlated time courses and ongoing dynamics of spontaneous activity also reflect other properties of neuronal networks. Following the intuition promoted here, spontaneous activity is determined, or at the very least constrained, by anatomical and physiological properties. Therefore, it is possible that spontaneous activity will conform to certain types of dynamics permissible by neural tissue. As noted above, several investigations have reported topographically organized spontaneous activity patterns that recapitulate sensory maps across the cortical surface. A more striking question is whether spontaneous neural activity at the spiking/ensemble level, e.g., within an orientation column of visual cortex, displays dynamical motifs of temporal behavior. This question has been addressed in a series of important studies by Yuste and colleagues, who utilized calcium-imaging methods to record and compare ensemble-spiking activity during spontaneous and evoked states [bib_ref] Attractor dynamics of network UP states in the neocortex, Cossart [/bib_ref] [bib_ref] Internal dynamics determine the cortical response to thalamic stimulation, Maclean [/bib_ref] [bib_ref] Visual stimuli recruit intrinsically generated cortical ensembles, Miller [/bib_ref] [bib_ref] Endogenous sequential cortical activity evoked by visual stimuli, Carrillo-Reid [/bib_ref]. A foundational question for quantifying structures in spontaneous ensemble activity is whether ongoing spiking displays repeating patterns of ensemble activity. Using twophoton calcium imaging, [bib_ref] Attractor dynamics of network UP states in the neocortex, Cossart [/bib_ref] studied the patterns of ensemble spiking in slice preparations of rodent visual cortex. During periods of spontaneous upstates, the authors observed repeating stereotyped ensemble activity, which reflected a "core" of regularly activated neurons. Given these stereotyped responses during spontaneous states, it is of interest to compare how the responses of single neurons relate to functional activation of the ensemble. To study this question, [bib_ref] Internal dynamics determine the cortical response to thalamic stimulation, Maclean [/bib_ref] employed two-photon calcium imaging in thalamocortical slices that covered somatosensory cortex. Using this preparation, the authors were able to quantify ensemblespiking activity triggered by both spontaneous activity and thalamic stimulation. Following previous observations, [bib_ref] Internal dynamics determine the cortical response to thalamic stimulation, Maclean [/bib_ref] identified consistent ensemble patterns of activity during spontaneous upstates. Stereotyped patterns of ensemble activity in somatosensory cortex were also observed during activation via electrical stimulation of the ventral thalamus, which also reflected a local depolarizing upstate. Strikingly, [bib_ref] Internal dynamics determine the cortical response to thalamic stimulation, Maclean [/bib_ref] observed no discernable difference between the ensemble patterns identified during spontaneous or thalamic triggered upstates, which was confirmed electrophysiologically via intracellular recordings of target cells. This similarity between conditions more generally supports the conclusion that spontaneous dynamics were not driven by thalamic inputs, as tested with this preparation. These observations lead the authors to speculate that thalamic feed-forward excitation works to release dynamic "trajectories" of activity that are intrinsic to cortex [bib_ref] Internal dynamics determine the cortical response to thalamic stimulation, Maclean [/bib_ref]. Again, this conclusion suggests that spontaneous dynamics follow patterns of network activity that conform to the predominant causal pathways of circuit interaction. Such claims, however, are limited by the fact that in vitro sampling of network activity lacks many aspects of neural activity in vivo, such as cortico-cortical feedback and neuromodulation from nuclei in the brain stem. More recently, [bib_ref] Visual stimuli recruit intrinsically generated cortical ensembles, Miller [/bib_ref] addressed this technical limitation by studying ensemble population activity using twophoton calcium imaging of visual cortex in awake rodents during spontaneous and visually stimulated conditions. These experimental conditions provide important challenges for previous observations, as the in vivo setting presents a far richer neural context of causal influence, and therefore the possibility for divergence of ensemble activity patterns between spontaneous and task evoked states. Comparing spontaneous periods to the presentation of visual gratings and naturalistic movies, [bib_ref] Visual stimuli recruit intrinsically generated cortical ensembles, Miller [/bib_ref] observed that the same ensembles of neurons were coactivated across conditions. This experimental preparation did not include electrophysiological recordings, and so explicit temporal similarity of spiking activity was not captured. However, consistent with the slice work reviewed above, these observations suggest that coactivation of a "core" ensemble of neurons is key to the intrinsic functional state of a cortical region. Indeed, [bib_ref] Visual stimuli recruit intrinsically generated cortical ensembles, Miller [/bib_ref] speculate that evoked functional responses sample from a "lexicon" of intrinsic functional ensemble configurations that are transiently expressed in spontaneous activity, in the absence of sensory events. Importantly, while this work focused on the identification of spatial ensembles, recent analysis of these data, focused on timeresolved extraction of ensemble similarities, further supports the view that canonical sequences of ensemble engagement during visual stimulation also occur during spontaneous states [bib_ref] Endogenous sequential cortical activity evoked by visual stimuli, Carrillo-Reid [/bib_ref]. Endogenous patterns of neural activity therefore provide a strong prediction of the possibility space of functional states during stimulation, even under conditions where animals are naïve to the experimental stimuli-excluding related phenomena such as preplay or replay of firing sequences [bib_ref] Endogenous sequential cortical activity evoked by visual stimuli, Carrillo-Reid [/bib_ref]. The hypothesis that neuronal task and spontaneous dynamics explore a common state space requires further support from studies with high-temporal resolution. Using tetrode recordings from rodent primary auditory cortex, [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] studied similarities of spontaneous and sensory-evoked population spiking activity. The authors first characterized the responses of neurons to tones and naturalistic stimuli. Although neurons differed in their stimulus-triggered spiking responses, each isolated neuron displayed stereotyped consistency of spiking activity for tones of differing frequency and naturalistic stimuli. Next, [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] studied the spiking activity of the same neurons during spontaneous upstate periods. Strikingly, the authors observed a strong correspondence between the stereotyped responses of a given neuron across tones, naturalistic sounds and spontaneous upstates. Importantly, the authors also confirmed that this similarity between evoked and spontaneous events is observed regardless of whether the animals are awake or anesthetized. [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] interpret the similarity of spiking sequences across states to reflect a set of constraints imposed on the population that greatly limit the repertoire of neural responses. To further explore this conjecture, the authors studied population spiking as multi-dimensional vectors of timeintegrated firing rates for each recorded neuron. In considering the simplest case of two neurons, [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] observed a strong overlap in responses of the two neurons during evoked auditory stimuli and spontaneous events, whereby spontaneous events occupied a large, structured 2-dimensional space that included the evoked events. Importantly, spontaneous activity did not occupy all of the response space uniformly, and was geometrically different from the space occupied by randomly shuffled data. This observation was extended to a multi-dimensional space across multiple neurons. When visualized in a 2-dimensional projection, the population vectors of spiking responses for different stimuli all occupied small clusters within the space formed by spontaneous activity, which itself was topologically distinct from randomly shuffled data. From this observation, the authors inferred that spontaneous activity explores and therefore conveys the boundary conditions of population dynamics, which form the shared constraints imposed on evoked responses. Given these limits, the authors suggest that functional responses of a local circuit population are constituted by a limited set, or "vocabulary, " of activity patterns, from within a neural response space outlined by spontaneous neural dynamics. What might be the sources of these shared constraints? A classical interpretation is to view local circuits as physioanatomic ensembles sculpted through maturation by shared statistical history of activation [bib_ref] Cognit activation: a mechanism enabling temporal integration in working memory, Fuster [/bib_ref]. Consistent with the general intuition advocated in this review, the Hebbian formation of local and global cell assemblies suggests that these spatio-temporal attributes, or constraints, will be expressed in ongoing spontaneous activity. Partial empirical support for this view, where ontogenetic and environmental factors sculpt circuit formation, is derived from work reviewed earlier, which shows that the similarity of topographic patterns in visual cortex during spontaneous and evoked states increases across maturation [bib_ref] Small modulation of ongoing cortical dynamics by sensory input during natural vision, Fiser [/bib_ref] [bib_ref] Spontaneous cortical activity reveals hallmarks of an optimal internal model of the..., Berkes [/bib_ref]. As discussed below, acute changes in neural dynamics, such as ensemble firing sequences, can occur via learning. These findings, combined, lead to interesting predictions about the factors influencing the similarity between spontaneous and evoked activity patterns, and the possibility of intervening in circuit organization to compare the reconfiguration of population dynamics during spontaneous and stimulated events. ## Preplay/replay spike sequences The electrophysiological and optical imaging data reviewed above suggest that neural populations display patterns of spontaneous activity that relate closely to evoked responses. This relationship suggests that using appropriate multivariate methods, functional response patterns of neuronal populations can be predicted from the statistical attributes observed during spontaneous non-task states [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] [bib_ref] Diverse coupling of neurons to populations in sensory cortex, Okun [/bib_ref]. How might these observations from sensory cortical regions relate to similar phenomena of spike sequence preplay and replay in the hippocampus? Place cells within the hippocampal subfields display spatial receptive fields, which modulate their firing rate as the animal traverses a preferred topological location (i.e., a place field). Recording multiple place cells with overlapping receptive fields will therefore display a progressive sequence of spiking activity as the animal moves through each successive place field [bib_ref] Prediction, sequences and the hippocampus, Lisman [/bib_ref]. Strikingly, it has been repeatedly observed that both prior to, and after, a learned locomotor trajectory is taken, the unique firing sequence across mapped placed cells is repeated [bib_ref] Forward and reverse hippocampal place-cell sequences during ripples, Diba [/bib_ref] [bib_ref] Internally generated cell assembly sequences in the rat hippocampus, Pastalkova [/bib_ref]. For both preplay (repeated spike sequence prior to locomotion) and replay (repeated spike sequence post locomotion), firing patterns are triggered by transient high-frequency activity called "ripples" [bib_ref] Hippocampal sharp wave-ripple: a cognitive biomarker for episodic memory and planning, Buzsáki [/bib_ref]. Interestingly, these spiking sequences can run in the forward direction, matching locomotion sequence, or backward direction [bib_ref] Forward and reverse hippocampal place-cell sequences during ripples, Diba [/bib_ref] , with the proportion of forward/backward sequences modulated by behavioral state [bib_ref] The balance of forward and backward hippocampal sequences shifts across behavioral states, Wikenheiser [/bib_ref]. More recent experimental work has shown that preplay sequences not only reflect learned sequences, but also express sequences reflecting novel trajectories of navigation and planned behavior [bib_ref] Decoding the cognitive map: ensemble hippocampal sequences and decision making, Wikenheiser [/bib_ref] ; but see [bib_ref] Trajectory events across hippocampal place cells require previous experience, Silva [/bib_ref]. Given that these events happen outside of explicit behavioral events, to what extent do they mirror the relationship between spontaneous and evoked activity patterns? Preplay and replay spike sequences differ in their relationship to task performance. While these sequences do occur during passive states, these states occur within a specific temporal context of task performance. In this fashion, they likely reflect a clear behavioral purpose related to the consolidation and learning of behavior, as well as to support planned action and decision-making. Making similar inferences about spontaneous data is difficult given the lack of explicit behavioral controls. However, preplay/replay phenomena in the hippocampus may be thought of as a well-documented case of how ensemble dynamics are strongly constrained by anatomical and biophysical factors, whereby local excitable events (e.g., ripples) trigger a limited set of spiking activity. In the case of preplay/replay, recent learning, and therefore activation of specific firing sequences, will bias the population to an even smaller response space when subject to excitatory drive. These general predictions suggest spontaneous activity may be biased by recent modulations of ensemble physiology, following a Hebbian rule, and that specific sequences can be released through acute learning and controlled excitation [bib_ref] Spontaneous events outline the realm of possible sensory responses in neocortical populations, Luczak [/bib_ref] [bib_ref] Stimulusinduced visual cortical networks are recapitulated by spontaneous local and interareal synchronization, Lewis [/bib_ref]. Consistent with this prediction, [bib_ref] Activity recall in a visual cortical ensemble, Xu [/bib_ref] have previously shown that V1 ensemble firing sequences to a learned trajectory of a visual target can be reproduced by presenting a transient visual stimulus at the starting location of the sequence. ## Future directions As reviewed above, spontaneous neural dynamics at multiple scales conform to basic anatomical and physiological constraints. Given this relationship, quantitative consideration of spontaneous activity patterns can provide provisional insight into the anatomical and dynamical properties of neural networks at the micro-, meso-, and macro scale. However, these inferences need to be made cautiously, as the measurement of neural activity is often confined to a limited spatial and temporal scale, and each measurement technique is biased to detect different domains of the underlying neuronal circuit functions. In addition, correlation and other statistical treatments of spontaneous data provide uncertain descriptions of underlying causal interaction. As briefly discussed below, these challenges can be addressed through improved statistical methods and causal experimental manipulations. ## Correlation, connectivity, and causal inference The challenges posed by the complexity of brain data are pervasive in neuroscience, and are particularly salient when investigating the neuronal basis of spontaneous brain activity across multiple scales. In many experimental settings, only restricted observations of interacting neural elements (e.g., cells/circuits/regions) can be achieved, often without the ability to causally intervene. These technical limitations highlight two important constraints of correlational methods for evaluating spontaneous brain activity. First, incomplete measurements of neural interactions limit the inferential power of observed correlations, as hidden factors may shape covariance. Indeed, a wide-ranging set of causal interactions can explain the same correlational structure. For example, regions A and B may exhibit correlated activity because they are directly connected, but this can also arise because of an indirect connection from A to B via a third region C, or from a common feed forward source, such as when C projects to both A and B. These alternative scenarios cannot be distinguished using correlation-based data analysis alone (when C is not observed). Thus, although at an aggregate level, rsfMRI covariance tends to follow anatomical connectivity, it does present some departure from tractographic connectivity estimates [bib_ref] Predicting human resting-state functional connectivity from structural connectivity, Honey [/bib_ref]. Secondly, correlation quantifies an undirected noncausal relationship between two variables. For example, correlations between two brain structures, A and B, may reflect a unidirectional or bidirectional causal interaction, or alternatively reflect no causal influence between elements, but rather a shared causal input from C. In this respect, causal information is essential to elucidating the actual structure of functional network topology. Ideally, casual inference is made through carefully controlled intervention of network/element interaction. The precision required for causal experimentation is often limited to invasive animal model systems, and some rare settings in humans [bib_ref] Intrinsic functional architecture predicts electrically evoked responses in the human brain, Keller [/bib_ref] [bib_ref] Corticocortical evoked potentials reveal projectors and integrators in human brain networks, Keller [/bib_ref]. However, there are several analytical methods that can be employed to partially deal with the limitations of the correlational approach. To address the degeneracy of coupling models that account for neural observations, it is necessary to employ methods that leverage additional assumptions [bib_ref] Improved estimation and interpretation of correlations in neural circuits, Yatsenko [/bib_ref]. Going beyond simple bivariate covariance measures, available methods range from relatively simple inverse covariance models [bib_ref] Network modelling methods for FMRI, Smith [/bib_ref] , auto-regressive causal models [bib_ref] Granger causality analysis in neuroscience and neuroimaging, Seth [/bib_ref] , Bayesian networks [bib_ref] Bayesian networks for fMRI: a primer, Mumford [/bib_ref] , laggedcoordinate embedding approaches [bib_ref] Detecting causality in complex ecosystems, Sugihara [/bib_ref] , as well as brain-specific approaches such as dynamic causal modeling [bib_ref] Dynamic causal modelling, Friston [/bib_ref] ; each trade off inferential power with added assumptions. Within many of these frameworks, assumptions of sparsity are used to mitigate the problem of false-positive connectivity that can arise from indirect influences [bib_ref] Causal network inference via group sparse regularization, Bolstad [/bib_ref]. Put more simply, it is generally assumed that a correlation matrix of measured elements likely overestimates the underlying statistical relationships. In turn, many data driven approaches seek to reduce complexity (dimensionality) by considering each pair-wise correlation relative to all other correlation pairs to help identify redundancy (e.g., partial correlation). Such approaches reflect a blind method for correcting overestimation, where correlation (regression) estimates are penalized (regularized) with respect to the remaining observed data [bib_ref] Regression shrinkage and selection via the Lasso, Tibshirani [/bib_ref]. However, with knowledge about the system under investigation and the statistical attributes of the measurement technique, more explicit forms of penalization can be applied to correlation matrices to eliminate false positives [bib_ref] Improved estimation and interpretation of correlations in neural circuits, Yatsenko [/bib_ref]. Improvements in these methods are becoming increasingly important as neuroscientific data sets grow exponentially in size. Finally, given the common use of linear methods, the influence of non-stationarity must be carefully considered, particularly given the growing focus on temporally resolved analyses [bib_ref] Dynamic functional connectivity: promise, issues, and interpretations, Hutchison [/bib_ref]. Although, analysis techniques can be used in cases where causal experiments are challenging, some progress has been made for invasively studying casual connectivity in the human brain. Using intracranial recordings (ECoG), [bib_ref] Intrinsic functional architecture predicts electrically evoked responses in the human brain, Keller [/bib_ref] measured causal interactions between recording sites using cortico-cortical evoked potentials (CCEPs). To measure CCEPs, cortex is stimulated at one cortical region (by running current between a pair of overlying electrodes) and stimulation-locked responses are measured at electrodes located at other sites on the cortical surface. [bib_ref] Intrinsic functional architecture predicts electrically evoked responses in the human brain, Keller [/bib_ref] measured resting BOLD functional connectivity in a group of six patients, and mapped CCEPs across the lateral cortical surface of the same individuals. They found that the magnitude of the CCEPs between sites was correlated with the strength of BOLD functional connectivity between those sites. This relationship between spontaneous BOLD correlations and CCEPs was specific to positive BOLD correlations; regions with anticorrelated BOLD signals (discussed below) showed unreliable CCEPs. In a follow-up study with a larger patient sample, [bib_ref] Corticocortical evoked potentials reveal projectors and integrators in human brain networks, Keller [/bib_ref] mapped the large-scale causal network composed of interregional CCEPs. They observed that, while short-range (<5 cm distance) causal links were often reciprocal, the reciprocity over longer distances (>5 cm) was no more than expected by chance, indicating that long-range connections are often unidirectional. Peri-rolandic circuits exhibited surprisingly widespread and powerful connections in these analyses, in contrast with their typically sparse BOLD correlations. Although causal stimulation analyses are still a developing field, findings of connection reciprocity and density constrain models of functional brain architectures. The ability of CCEPs to resolve the directionality of neural information flow in the human brain makes them crucial for understanding large-scale spontaneous neural dynamics [bib_ref] Studying network mechanisms using intracranial stimulation in epileptic patients, David [/bib_ref]. ## Anti-correlation? Electrophysiological measurements will assist us in moving beyond the question of whether two regions are functionally connected to a description of how two regions are interacting. More specifically, electrophysiological data will be crucial in transitioning from static to dynamic accounts of large-scale network configuration [bib_ref] Dynamic functional connectivity: promise, issues, and interpretations, Hutchison [/bib_ref]. For example, it is crucial to determine whether the information transmission between cortical regions is primarily feed forward (i.e., driving) or feedback (i.e., modulatory) in nature. Similarly, it is important to distinguish which inter-regional interactions are effectively excitatory and which are effectively inhibitory (suppressive). It has proven difficult to resolve these questions using functional neuroimaging. The literature harbors a decade of debate over whether functional antagonism can be inferred from BOLD anticorrelation (i.e., from the fact that increased BOLD signal in one site is associated with decreased BOLD signal at another site). On the one hand, the pattern of BOLD anti-correlations has been put forward as a powerful principle of large-scale neural organization [bib_ref] The human brain is intrinsically organized into dynamic, anticorrelated functional networks, Fox [/bib_ref] with clinical implications [bib_ref] Competition between functional brain networks mediates behavioral variability, Kelly [/bib_ref] [bib_ref] Resting-state networks link invasive and noninvasive brain stimulation across diverse psychiatric and..., Fox [/bib_ref]. On the other hand, BOLD fMRI anti-correlations may arise from artifacts of acquisition, physiology or signal processing [bib_ref] Effects of model-based physiological noise correction on default mode network anti-correlations and..., Chang [/bib_ref] [bib_ref] The impact of global signal regression on resting state correlations: are anti-correlated..., Murphy [/bib_ref] [bib_ref] Correlations and anticorrelations in resting-state functional connectivity MRI: a quantitative comparison of..., Weissenbacher [/bib_ref] [bib_ref] Trouble at rest: how correlation patterns and group differences become distorted after..., Saad [/bib_ref]. Electrophysiological investigations can help resolve uncertainties associated with interpreting BOLD anticorrelations. [bib_ref] Intrinsic functional architecture predicts electrically evoked responses in the human brain, Keller [/bib_ref] observed that positive BOLD correlations were consistently associated with causal CCEPs, while negative BOLD correlations were not. Subsequently, [bib_ref] Neurophysiological investigation of spontaneous correlated and anticorrelated fluctuations of the BOLD signal, Keller [/bib_ref] observed that, while most negative BOLD correlations do not correspond to negative correlations in electrophysiologically measured population activity, a subset of BOLD anti-correlations matches anti-correlations of 50-150 Hz power measured electrophysiologically. Thus, a minority of anticorrelated rsfMRI networks may reflect suppressive interaction at the population level. In addition, electrophysiological measures allow us to resolve how rsfMRI correlations vary over time, which may reveal further antagonistic relationships. For example, a link with zero aggregate correlation may in fact be due to rapid switching between antagonistic and supportive modes of interactions. Although the interpretation of BOLD anti-correlations remains difficult, it is clear that electrophysiological methods with high temporal resolution and broad fields of view will be important in resolving these and related questions. Importantly, under task conditions, human intracranial data provides strong support for dynamic anticorrelated activity. For example, [bib_ref] Transient suppression of broadband gamma power in the defaultmode network is correlated..., Ossandón [/bib_ref] reported brain-wide anti-correlation patterns in human intracranial recordings closely matching prior fMRI findings. [bib_ref] Transient suppression of broadband gamma power in the defaultmode network is correlated..., Ossandón [/bib_ref] observed these anti-correlations in broadband 60-140 Hz activity during an active visual search task. Such data provide a clear electrophysiological basis for the correlation and anti-correlation patterns typically seen with fMRI within and between the "task-positive" and "task-negative" networks. ## Pathological spontaneous activity There is now a large literature that applies rsfMRI network analyses to different neurological and psychiatric patient populations. This literature has been extensively reviewed elsewhere [bib_ref] The role of impaired neuronal communication in neurological disorders, He [/bib_ref] [bib_ref] Clinical applications of resting state functional connectivity, Fox [/bib_ref] [bib_ref] Disease and the brain's dark energy, Zhang [/bib_ref] [bib_ref] The connectomics of brain disorders, Fornito [/bib_ref]. Importantly, while neurological disease is often typified by explicit brain pathology and injury, such explicit causes have not been as readily identified in psychiatric illness. Rather, psychiatric illness can be viewed as perturbations in the functional organization or temporal dynamics of otherwise superficially healthy neural tissue [bib_ref] Human brain networks in health and disease, Bassett [/bib_ref]. Clinically, this presents a challenge for identifying pathological network structure and dynamics as biomarkers for treatment. Initial applications of rsfMRI have provided promising results in psychiatric disorders ranging from schizophrenia [bib_ref] Altered global brain signal in schizophrenia, Yang [/bib_ref] and autism [bib_ref] The perils of global signal regression for group comparisons: a case study..., Gotts [/bib_ref] to depression [bib_ref] Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory..., Ressler [/bib_ref]. In addition, a bold, contemporary initiative seeks to elucidate the functional-anatomical markers that correlate with emotional, perceptual and cognitive symptoms common to different psychiatric disorders, and to use these neural correlates as therapeutic targets [bib_ref] Next-generation treatments for mental disorders, Insel [/bib_ref] [bib_ref] Resting-state networks link invasive and noninvasive brain stimulation across diverse psychiatric and..., Fox [/bib_ref]. As part of this endeavor, consideration of spontaneous neural dynamics can help contribute to functional brain network identification and the quantification of underlying differences in network organization across psychiatric conditions [bib_ref] The connectomics of brain disorders, Fornito [/bib_ref]. Studies focused on spontaneous dynamics may also help support alternative accounts of differences in functional network dynamics associated with psychiatric disease (e.g., . # Conclusion Evidence from multiple scales of neural organization suggests that consideration of spontaneous neural dynamics can provide insight into the basic organization of functional neural systems. Importantly, the recent interest in rsfMRI investigation of largescale brain networks is supported by a clear correspondence to invasively recorded neural activity in humans and nonhuman primates. These invasive recordings have further revealed important neural organizational principles at the mesoscopic and microscopic scales. As we gather more information about the electrophysiological basis of large-scale neural connectivity and dynamics, a major goal should be moving from descriptive to more mechanistic models of neural dynamics and network organization. This progress is a necessary step toward harnessing the power of spontaneous brain dynamics for more effective treatments of psychiatric and neurological disorders. [fig] FIGURE 1 |: Quantifying band limited amplitude for correlation analysis of spontaneous data. (A) Raw spontaneous ECoG time series (5 s). (B) Spontaneous alpha band limited time series extracted from (A), by a 7-10 Hz band pass filter. (C) Analytic signal of (B), obtained via a Hilbert transform, showing the real component (blue), imaginary component (red), and amplitude/envelope (green). The real component is the original band pass signal from (B), and the imaginary component is a 90 • rotated instantiation of (B). The amplitude/envelope (green) is the absolute value of the analytic signal (i.e., a complex valued time series with real and imaginary parts). (D) Complex plane showing the temporal evolution (yellow-green shading) of the analytic signal [data comes from window highlighted with gray in (C)]. Over time, each observation (sample) takes a coordinate location in the complex plane given the real (x-axis) and imaginary (y-axis) values at that time point [example time point with white fill color highlighted in (D,E)]. For any given time point, the amplitude/envelope of the signal in the complex plane is defined by the vector length extending from the central zero axes to the real/imaginary coordinate (red line). For example, as time evolves in (D; progress toward dark green) the trajectory of values spirals out radially, increasing the vector distance from the origin: this reflects an increase in alpha band amplitude as shown in (E). Phase can also be obtained by identifying the angular position of the amplitude vector at each time point (red vector shown has a phase angle of 30 • ). [/fig]

Allosteric Recognition of Homomeric and Heteromeric Pairs of Monosaccharides by a Foldamer Capsule The recognition of either homomeric or heteromeric pairs of pentoses in an aromatic oligoamide double helical foldamer capsule was evidenced by circular dichroism (CD), NMR spectroscopy, and X-ray crystallography.T he cavity of the host was predicted to be large enough to accommodate simultaneously two xylose molecules and to form a1 :2 complex (one container,t wo saccharides). Solution and solid-state data revealed the selective recognition of the a-4 C 1 -d-xylopyranose tautomer,w hichi sb ound at two identical sites in the foldamer cavity.Astep further was achieved by sequestering ah eteromeric pair of pentoses,t hat is,o ne molecule of a-4 C 1 -d-xylopyranose and one molecule of b-1 C 4 -d-arabinopyranose despite the symmetrical nature of the host and despite the similarity of the guests.Subtle inducedfit and allosteric effects are responsible for the outstanding selectivities observed. # Introduction Thed evelopment of selective saccharide receptors is anotoriously difficult endeavor, so much so that few research groups dare challenging it. Saccharides nevertheless con-stitute ac entral class of biomolecules,a nd their chemical synthesis is an important subfield of organic chemistry. Discriminating,sensing,and selectively manipulating saccharides in water and in organic solvents thus remain subjects of broad interest, and also provide genuine opportunities to push forward the boundaries of molecular recognition. At the notable exception of receptors for all equatorial sugars, the ab initio design of selective saccharide receptors has not been achieved. Screening therefore remains acommon method. A typical approach has consisted of shaping binding sites from first principles and then screening which sugar binds best. Thus,v arious families of receptors,s uch as macrocycles, tripods,self-assembled metallo-organic capsules,and helically folded containers, have been produced, which often showed good affinity,a nd some selectivity,i ncluding for saccharides other than glucose derivatives. Systematic variations of the receptor structure may then permit improvements of binding selectivity and affinity. In the case of aromatic amide helical foldamers, conformations are predictable through energy minimization, allowing for the design of the cavity volume and the positioning of binding features.W hen the helix has ar educed diameter at both extremities,i ts urrounds its guest and secludes it from the solvent. Guest binding and release then require al ocal unfolding. Because of their folding mode, such capsules are relatively rigid in all kinds of solvents and therefore operate as size and shape selectors:i tw as for example possible to bind selectively ad ipentose at the exclusion of dihexoses,w hich were too large to fit into the cavity. Furthermore,t heir modular nature provides quick access to structural variants,u sing ac ommon synthetic scheme to add, delete,orm utate monomers. An advance brought by aromatic-foldamer-based saccharide receptors was straightforward access to detailed structural elucidation, including the very first characterization of complexes by single-crystal X-ray diffraction at atomic resolution. Crystal growth was facilitated by the rigid nature of the foldamer helices and by the use of racemic crystallography,t hrough mixing the racemic sugar with the racemic host. Based on this structural information, we showed that it is possible to iteratively design asugar receptor, that is, to introduce precise modifications so as to enhance selectivity in just afew rounds.Negative design, that is,the preservation of ab inding mode to ag iven guest and the rational introduction of modifications to exclude all other guests was demonstrated. Therational reversal of guest selectivity was also achieved, using two guests that differ by asingle hydroxy group. Encouraged by this background, we endeavored to develop ar eceptor able to bind two different monosaccharides simultaneously.T he study of bi-and multimolecular recognition has made it possible to explore new forms of stereoisomerism,to perform chemical reactions in confined spaces,and to construct supramolecular switches and logic gates.Its extension to carbohydrates was initially intended as acuriosity-driven molecular recognition challenge and also as amilestone towards receptor-mediated selective reactions between unprotected saccharides.Asshown in the following, our attempt was successful and also proved to be rich with several important discoveries and lessons.First, the shape and selectivity filter of aromatic amide capsules for saccharide binding is shown to be recurrently effective.Selective binding was not an objective of the current study but an essential result:the few guests that have been tested have aprevailing binding mode and are thus in principle amenable to structurebased rational iterative improvements. In one case,o ne complex out of 42 possible host-guest combinations selectively forms.Second, heteromeric saccharide recognition was found to prevail by simultaneously binding a-4 C 1 -d-xylopyranose and b-1 C 4 -d-arabinopyranose despite the C 2 symmetry of the host, ar ather counterintuitive process.H eteromeric guest binding in symmetrical hosts has been implemented before through space filling:when one guest fills more than half the available space,asecond, smaller guest is still allowed in the remaining space.H owever,t he mechanism here is different and seems to proceed via as ubtle allostery.T hird, induced fit and allostery are responsible for the occupation of different binding sites by the same guest depending on whether or not another guest is present. Alternatively,t hey may prevent the binding of ah omomeric pair of ag uest but nevertheless can form ah eteromeric pair. Thei ntriguing equilibria shown in , including the unusual substitution of one of two identical guests by ad ifferent molecule, schematize these findings. # Results and discussion Design, Synthesis, and Characterization of aDouble Helical Capsule Taking advantage of the predictability of aromatic oligoamide foldamer structures,w ep reviously designed unimolecular capsule 1, which proved to be efficient at stereoselectively binding small organic acids. In 1,t he quinoline trimers at each extremity close the helix cavity and also prevent its self-assembly into multiple helices. Indeed, high helix curvature,a si nq uinolinecarboxamide oligomers,d isfavors the spring-like extension associated with double helix formation.We envisioned that the removal of these trimers would allow the strands to form as table double helical architecture endowed with as ignifi- . Schematic representationo fthe reported 1:1and 1:2hostguest complexes formed from adouble helical host and two monosaccharides (red or blue spheres). Top: in the absence of guest, the host is filled by water molecules or solvent (small purple spheres). The blue guest forms a1:1 complex and cannot form a1:2 complex alone, but it does in the presence of the red guest. Note that the blue guest occupies different binding sites in the 1:1and heteromeric 1:2 complexes. cantly larger cavity than asingle helical analogue. Oligomer 2 was designed based on this assumption. Its sequence is am odified version of 1 in which the quinoline segments have been replaced by pyridinecarboxamide dimers. Thec onvergent synthesis of 2 involves the coupling of ap ivaloyl-PP A mono-acid with the amine of H 2 N-PN 2 -Boc using PyBOP as the coupling reagent. After Boc cleavage, pivaloyl-P 3 N 2 -NH 2 was coupled twice to the diacid of pyr-pyzpyr to provide 2 (see the Supporting Information). Thechoice of as elf-assembled receptor aimed to simplify the synthesis. Thed ifferent blocks mentioned above can be prepared on multigram scales,allowing us to readily obtain large amounts (> 1g)of2. Thes olid-state crystallographic structure of 2 was solved and revealed a2nm long duplex in which each strand spans three helical turns. Thet wo strands are helically offset with respect to one another by half at urn and extensively stack on top of each other. No other obvious interstrand interactions were noted. Thed uplex has three pseudo-C 2 symmetry axes,one along the helix axis and two in orthogonal directions. Evidence of double helix formation was also found in solution. The 1 HNMR spectrum of 2 recorded at 2mm in CDCl 3shows slightly broadened peaks.T he aromatic amide resonances appear in the 10-8.5 ppm region, at significantly higher field than what is usually found in single helical aryl amide capsules,ahallmark of double helix formation in these systems.Diluting down to 0.05 mm did not allow for the detection of single helix resonances in this solvent. However,t he addition of [D 6 ]DMSO, ac ompetitive solvent that disfavors double helix formation, led to the emergence of asecond set of sharp signals at lower fields,w hich were assigned to the single helix. In pure [D 6 ]DMSO,o nly the single helix is observed. Indeed, the structure of ac rystal grown from DMSO was solved and shown to be the single helix. In CDCl 3 /[D 6 ]DMSO (9:1 v/v), the single helix can be detected as am inor species at 180 mm.I ntegration of the signals provided am inimal estimate of the dimerization constant as K dim = 4 10 5 m À1 ,avalue large enough to consider (2) 2 to be asingle entity at the concentrations used in this work. ## Prediction of polar guest binding At rend has emerged from the host-guest properties of various aromatic amide foldamer capsules studied in independent contexts:tight and selective binding goes along with an occupancyofthe host cavity volume by the guest of at least 70 %. Guests that are smaller than optimal also bind but with al ower affinity.G uests that are too large for the available space do not bind at all. Another aspect to consider is aw eak but non negligible ability of the host to adjust its conformation to the volume of the guest through slight changes in helix curvature,t he pitch remaining constant and equal to the thickness of one aromatic ring. In the few cases where the structure of the host has been elucidated in the absence of guest, that is,w hen the host is filled with solvent only,t he cavity was found to be slightly smaller than in the presence of ag uest. Thef olded duplex structure (2) 2 has ap olar cavity,a se videnced by the presence of ten crystallographically defined water molecules, and an inner volume of 280 3a, e, f). We surmised that this cavity may be large enough to harbor two aldopentose guests. Xylose has av olume of 107 3 ,a nd two molecules of xylose would occupy 77 %o ft he cavity volume measured in the absence of guest, and presumably as maller fraction of the space available in an actual host-guest complex. In contrast, the volume of ahexose (ca. 130 3 )i sc learly too large to fit twice in the cavity of (2) 2 .W et herefore concentrated our efforts on the recognition of pentoses. ## Solution and solid-state study of d-xylose encapsulation Thea bility of (2) 2 to bind pentoses was assessed by titrations in CHCl 3 /DMSO (9:1 v/v) at 298 K. Ac ircular dichroism (CD) titration of achiral (2) 2 with d-3 showed the appearance of an egative induced CD signalresulting from helix handedness bias.T he changes in ellipticity could be fitted to a1 :2 binding model, which afforded an overall binding constant K = 2.19 10 9 m À2 corresponding to K a values of 69 200 m À1 and 31 600 m À1 for the binding of af irst and second d-xylose guest, respectively. These values reflect aslightly positive cooperativity (a = 4K a2 / K a1 = 1.8), illustrated by the sigmoidal binding isotherm.A 1 HNMR titration under the same conditions revealed the appearance of as ingle new set of sharp helix signals upon binding of d-3. Thenumber of amide resonances (12) is indicative of ap seudo-twofold symmetry,that is,alower symmetry than for the double helix in the absence of xylose.Inagreement with this result was the fact that the 1 H-13 CH SQC spectrum of encapsulated, uniformlysimplicity of the HSQC spectrum also reflect ac omplete selectivity for asingle tautomer of d-3,associated with the full diastereoselectivity for ag iven handedness of the double helix. By considering the 13 Cc hemical shifts,t he dihedral angles between CH and OH groups derived from 3 J coupling constants,a nd COSY and TOCSY two-dimensional NMR experiments,itwas possible to determine that the two guests adopt an a-4 C 1 -pyranose puckered conformation. Thes pontaneous occurrence of such alevel of selectivity is significant. Considering the a/b-anomers of the guest and the P/M-helicity of the host, six different 1:2c omplexes may form. Solution data not only show that one of them prevails, but also that one binding mode must prevail as well. Despite the many hydrogen bond donors and acceptors on both the guest and host, aparticular orientation is preferred. Further characterization of the complex was performed by 2D and 3D NMR spectroscopy. 1 H-1 HR OESY spectra recorded at 298 Kr evealed the existence of exchange crosspeaks between protons of the capsule strands,r eflecting the dynamic nature of the pseudo-C 2 -symmetrical complex in solution. Theexchange rate between the two populations was measured to be 4.9 AE 0.2 s À1 ,and is thus on the same timescale as that required by the multidimensional NMR spectra. Decreasing the temperature to 278 Ks uppressed the exchange. This allowed for extensive 1 H,C, andNc hemical shift assignments of the spectra of (2) 2 '(d-3) 2 (Tables S1-S3 and Figures S12 and S13) and the determination of ah igh-resolution NMR structure of the complex. A final ensemble of 20 structuresw as calculated from distance restraints measured on asample of 13 C-labeled d-3 bound to 13 C-natural abundance (2) 2 .T he use of 13 Cedited and -filtered NMR spectra allowed for the collection of 334 unique distance restraintsand , including 82 intermolecular restraints to accurately position the monosaccharides within the capsule cavity and 190 interand intrastrand restraints to position the strands relative to one another, as well as the side chains.T he structure also revealed the presence of two bound water molecules located at the extremities of the double helix and the assignment of P-helix handedness.I nt he complex, the two sugar-binding sites are identical and consist of the two inequivalent extremities of the two strands.T he dynamic exchange mentioned above can thus be assigned to as liding motion of the two strandswith respect to one another, along with ac oncomitant repositioning of each sugar within its cavity. In parallel, the crystal structure of racemic (2) 2 'd/l -3 was elucidated. Theu se of racemic crystallography recently helped delivering the very first structures of receptor-sugar complexes, but crystal growth is also ac onsequence of the complex structure being well defined in solution. There is an ear-perfect superposition of the ensemble of solution structures with the solid-state crystallographic analysis with aroot mean square deviation (rmsd) of 0.054 AE 0.03. Them atch includes the pseudo-C 2 symmetry,t he presence and position of the two sugars,a nd the presence of the two water molecules,a sw ell as the P-handedness of the double helix containing d-3.T he solidstate structure also confirmed the a-4 C 1 -pyranose tautomeric form of the sugars.T he centrosymmetric P-1 space group implies that the crystal lattice also contains two a-1 C 4 -lxylopyranose molecules encapsulated by M-(2) 2 .T he segregation of d-and l-3 in P-and M-helices,respectively,isinline with the full diastereoselectivity observed in solution. Thes ugar racemate could also yield ad iastereomeric heterocomplex including both d-3 and l-3.H owever, the latter did not crystallize and was also not observed in solution. Fore xample,w hen a1 :1 (pseudo-racemic) mixture of 13 C-labeled d-3 and 13 C-natural abundance l-3 was added to (2) 2 ,H SQC spectra confirmed that only one type of complex formed. In both the solution and solid-state structures,t wo water molecules were found to be encapsulated with the carbohydrates,each occupying an extremity of the cavity.These water molecules are held in position through multiple hydrogen bonds with the amide protons of the terminal pyridine monomer of each strand. Additionally,d irect water-tosaccharide hydrogen bonding is observed. Thes tructures show an extensive array of eight hydrogen bonds between each sugar hydroxy group and the inner wall of the helix and. No intramolecular hydrogen bonds were found between neighboring hydroxy groups of the monosaccharides.Only host-guest intermolecular hydrogen bonds exist. Four of the eight hydrogen bonds involve hydroxy proton donors and the other four involve amide proton donors.E ach sugar exposes its endocyclico xygen atom and methylene group to the center of the cavity, forbidding inter-sugar hydrogen bonds.T he weak positive binding cooperativity observed is thus mediated by the helix backbone,n ot by guest-guest interactions,d espite the fact that the guests do not break the symmetry of the host. Comparison of the solid-state structures of the empty host and of the (2) 2 '(d-3) 2 complex confirms that aconformational change takes place upon binding:t he two strands of (2) 2 are helically offset with respect to one another by aq uarter of aturn in the complex as opposed to half at urn in the empty capsule,revealing some kind of induced fit at one of the rare degrees of structural freedom of the duplex. As ar esult, the central monomers of each strand are at an angle of about 608 8 in the complex instead of being in front of each other in the empty capsule.C oncomitantly,t he inner volume increases from 280 to 306 3 upon guest binding.T his allowed us to calculate that 70 %o ft he volume is occupied by the two xylose molecules,aspredicted initially. ## Solution studies on d-arabinose binding Thebinding of d-arabinose 4,apentose that differs from d-3 by only two stereogenic centers,w as then evaluated. A titration of (2) 2 with d-4 in CHCl 3 /DMSO (9:1 v/v) at 298 K was monitored by CD spectroscopy.Apositive signal centered at 370 nm appeared upon increasing the concentration of d-4. TheC Di ntensity remained significantly weaker than with d-3.I na ddition, the band at 370 nm had an opposite sign but this did not apply to other bands,h inting at variations of the CD spectra not only through the handedness of the helix but possibly also through the relative positioning of the two strands.Inthe case of d-4, the changes in ellipticity were inconsistent with a1 :2 stoichiometry but instead fitted to a1 :1 binding model, which afforded a K a value of 21 900 m À1 ,alower affinity than for d-3.A 1 HNMR titration in CDCl 3 /[D 6 ]DMSO (9:1 v/v) at 298 Kw as attempted. However,a lthough spectral changes were clearly visible,t he signals were too broad to be interpreted, probably because of guest tumbling and/or the presence of disordered solvent molecules inside the cavity.The same experiment repeated at 243 Krevealed the emergence of reasonably sharp amide peaks.Asfor the CD titration, the spectrum does not change appreciably after addition of 1equiv of d-4.T he same titration was carried out with 13 C-labeled d-4 and monitored by 1 H-13 CHSQC spectroscopy.Again, addition of more than 1equiv of guest did not lead to any variation of the complex resonance pattern (highlighted in blue in. TheH SQC data revealed two distinct resonances for the anomeric C1 carbon atom of the 13 C-labeled d-4 encapsulated in (2) 2 .T hese cross-peaks have as imilarCc hemical shift, and are thus unlikely to correspond to different anomers of the sugar.I nstead, we assigned them to diastereoisomeric complexes of au nique anomer of d-4 being encapsulated either in P-o rM-(2) 2 .I ntegration of the two cross-peaks allowed the diastereomeric excess to be calculated to be 30 %, which is consistent with the lower CD intensity.Based on theCc hemical shifts,t he dihedral angles between CH and OH groups derived from 3 J coupling constants,a nd two-dimensional COSY and TOCSY NMR experiments,i tw as possible to determine that the guest is in a b-1 C 4 -pyranose puckered conformation. In this conformation, only one guest molecule is allowed in the double helix cavity. Thebinding of arabinose thus appears to be less selective than that of xylose.H owever,i tc an be inferred from the NMR data that the two complexes observed are well-defined, including the conformation of the sugar. Theabsence of a1:2 complex even though arabinose is not larger than xylose is also indicative of tight and selective interactions.A rabinose will not migrate to al ocation of the host cavity that would allow for asecond guest to bind. Thesize and shape selectivity filters of aromatic amide foldamer cavities evoked above are again at play. ## Structural and thermodynamic studies of the formation of aheterodimeric d-xylose-d-arabinose complex Next, we sought to evaluate whether aheteromeric pair of pentoses could be encapsulated by (2) 2 .CDmonitoring of the addition of d-4 to (2) 2 previously equilibrated with excess d-3 revealed that the initial spectrum, typical of (2) 2 '(d-3) 2 , changed to eventually reach afinal state suggesting some sort of saturation. Thef inal spectrum was very different from that of (2) 2 '(d-4), the expected final product if arabinose had simply replaced the two xylose molecules.This result hinted at the possible formation of heteromeric (2) 2 '-(d-3;d-4). Thechanges in ellipticity could indeed be fitted to such ap rocesst oa fford a K a value of 46 800 m À1 for the equilibrium (2) 2 'd-3 + d-4Ð(2) 2 '(d-3,d-4). In other words, d-4 has an affinity for (2) 2 'd-3 that is more than twice as large as that for (2) 2 .C onversely,( 2) 2 'd-3 has as lightly larger affinity for d-4 than for d-3. Monitoring the same titration by 1 HN MR spectroscopy revealed the emergence of anew set of peaks as d-4 probably replaces one of the d-3 gueststo form ah eteromeric complex. Consistent with this interpretation, the number of the capsule amide peaks was doubled relative to what was found for the symmetrical (2) 2 '(d-3) 2 ,i mplying that the final structure had no symmetry at all. Titrations with 13 C-labeled d-3 and d-4 monitored by 1 H-13 CH SQC spectroscopy eventually provided unequivocal evidence for heterocomplex formation. Upon adding d-4,t wo new cross-peaks appeared that correspond to the encapsulated C1 anomeric carbon atoms of both d-3 and d-4.T he unambiguous assignment of the sugar resonances was achieved by using 13 C-labeled d-3 in the presence of unlabeled d-4 and vice versa . We also found that increasing the temperature to 318 Ki ncreased the proportion of the heterocomplex to more than 90 %. This led us to study the effect of temperature on the replacement of d-3 by d-4,w hich can be described by the equilibrium:( 2) 2 '(d-3) 2 + d-4Ð(2) 2 '(d-3;d-4) + d-3.T he linear vantH off plots showed that the process is enthalpically disfavored and entropy-driven (DH = 32 kJ mol À1 ; DS = 0.11 kJ mol À1 K À1 ,i .e., ÀTDS = À32.8 kJ mol À1 at 298 K): below room temperature homocomplex formation is favored while above it heterocomplex formation prevails.T he origin of such alarge entropic component for asubstitution process is unclear and may be related to the release of encapsulated water molecules. Yet, no such effects have been observed in the other equilibria investigated here or in our earlier studies on foldamer-saccharide recognition. Detailed structural information could not be gathered using crystallography.However,advanced NMR spectroscopic techniques allowed for the structural determination of the complex. As in the case of (2) 2 '(d-3) 2 ,exchange between the two strands of the capsule hampered the resolution of the structure at 298 K. Upon cooling down to 278 K, the exchange signals disappeared, but the amount of heterocomplex present in solution dropped because of the large entropic term mentioned above.N evertheless,t he concentration of (2) 2 '(d-3;d-4)remained sufficient for apartial assignment of the resonances and. By using combinations of natural-abundance and 13 C-labeled sugars it was eventually possible to determine an NMR structure of the complex. Af inal ensemble of 15 structuresw as calculated from at otal of 54 distance restraints,i ncluding 30 sugar-capsule intermolecular restraints that allowed us to position accurately both pentoses within the capsule cavity, and 24 inter-a nd intra-sugar restraints to determine the configurations and relative orientations of the guests. Thes tructure revealed that the arabinose adopts a b-1 C 4 -d-pyranose conformation whereas the xylose remains as an a-4 C 1 -d-pyranose. An array of eight hydrogen bonds between the hydroxy groups of each sugar and the inner wall of the helix hold the guests in place and. Thexylose position is identical to that of the homo-complex. Both pentoses are oriented with their endocyclic oxygen atom pointing to the same side of the cavity,leaving OH4 of d-3 relatively close to OH1 and OH2 of d-4 (3.4 ). Although it was not possible to confidently obtain distance restraints to accurately position the two water molecules located at the extremities of the complex, these were kept in the structure.T he inner volume of the cavity was found to be 325 3 ,and is slightly larger than that of the homo-complex. It thus appears that the replacement of one d-3 guest by d-4 can be mediated by subtle allosteric variations,b ut that the changes are important enough to prevent asecond substitution ((2) 2 '(d-4) 2 was not observed). Theh ost-mediated allosteric communication between two guests of identical size that differ by two stereogenic centers,x ylose and arabinose,w ithout contact between them appears to be unique.Usually,ahost meant to bind two different guests would be designed with two different binding sites,f or example,t ob ind an ion pair.Alternatively,symmetrical hosts have been shown to bind to heteromeric pairs of guests when the first guest occupies more than half of the space available,l eaving room only for asmaller guest.At last, we challenged the stereoselectivity of the complexation of both 3 and 4 by (2) 2 to draw ap arallel with the mutual exclusion of d-3 and l-3 mentioned above.T he addition of d-3 to (2) 2 '(d-4)f irst produced (2) 2 '(d-3;d-4) and then, with al arge excess of d-3,l ed to (2) 2 '(d-3) 2 .I n contrast, the addition of l-3 to (2) 2 '(d-4)did not produce any heteromeric complex. Instead, d-4 is replaced by l-3 to first produce (2) 2 '(l-3)a nd then (2) 2 '(l-3) 2 . Note that these competition experiments also involve some changes in helix handedness as (2) 2 '(d-3) 2 is P-w hereas (2) 2 '(l-3) 2 is M-helical. Assessing the selectivity of sugar binding through the screening of alarge number of different pentoses or hexoses was not the purpose of the present investigation. Yet, selectivity eventually turned out to be our main finding.T he theoretical outcome of mixing d-xylose and d-arabinose with ar acemic P/M-capsule is that no less than 42 different hostguest complexes may be produced. Theo bservation of as ingle heteromeric pair of sugars composed of a-4 C 1 -d-xylopyranose and b-1 C 4 -d-arabinopyranose in the P-helical foldamer cavity is thus outstanding. # Conclusion In conclusion, we have prepared adouble helical foldamer container with al arge internal cavity by using as trategy combining the folding and the self-assembly of ar eadily accessible aromatic oligoamide strand. Thecontainer stereoselectively encapsulates asingle homochiral pair of one xylose tautomer, a-4 C 1 -d-xylopyranose.W et hen demonstrated the fully selective complexation of aheteromeric pair of pentoses. Together with earlier studies,these results concur to show that aromatic amide helices act as stringent shape and selectivity filters for carbohydrate binding.I nr eference to the first sentence of the introduction about the challenge of carbohydrate recognition, it appears that general solutions are emerging.A no utcome of the formation of well-defined complexes is the possibility to accurately elucidate their structures and unravel the recognition, induced fit, and allosteric mechanism at play.Inturn, the obtained host-guest complex structures may constitute new starting points for structure-based iterative design and, eventually,t he further improvement (i.e., exclusion of all sugars but one) or even the reversal of guest selectivity. Fort his purpose,a dvanced predictive computational tools would bring am ajor advantage,and their development is highly needed. Our results also open up the possibility to precisely design confined spaces that could alter and control the reactivity of native carbohydrates and behave as molecular flasks. are highlighted in yellow and green, respectively.b)Enlarged side view of the cavity showing the heterocyclest hat interact with the guests and the water molecules. The heterocycles are color-coded in light gray or dark gray depending on the strand that they belong to. The hydrogen bonds found in the complex are shown as cyan dashed lines. Details of these hydrogen bonds can be found in the Supporting Information.c)Optimized structure of the complex, shown in thin tube representation for the host and in thick tube representation for the guests.E ach strand is colored in adifferent shade of gray.Non-polar hydrogen atoms, isobutoxys ide chains, and cavity-excluded solvent molecules have been removed for clarity. The volume of the cavity (325 3 )i sshown as atransparentpink isosurface. d) Formula and monomer numbering of each strand of the double helical capsule together with the structures of d-3 and d-4 shown as Mills projections.Hydrogen bonds where the sugars act as acceptors or donors are shown as red and blue dashed lines, respectively.R= isobutyl.

Post‐COVID‐19 fatal Aspergillus endocarditis: A case report ## | introduc ti on Aspergillus endocarditis (AE) has been reported in immunocompromised and immunocompetent patients with a history of open-heart surgery, valve replacement, or intravenous drug use. The high mortality rate among survivors is very important due to the risk of relapsing infection. [bib_ref] Aspergillus tubingensis endocarditis: a case report and review of the literature, Born [/bib_ref] A literature review from 2005 to 2016 showed that out of 374 cases of infective endocarditis, 43 (11.5%) had fungal infections, and the most common fungi (31/374 patients; 8.3%) were Aspergillus and Candida (9/374 patients; 2.4%). [bib_ref] Fatal prosthetic valve endocarditis due to Aspergillus flavus in a diabetic patient, Jalalian [/bib_ref] Extracardiac manifestations, mistaken/delayed AE diagnosis, and long duration of symptoms before going to the hospital may contribute to wrong management in patients then causing a delay in the treatment and conclusive poor outcomes. [bib_ref] Aspergillus endocarditis: diagnostic criteria and predictors of outcome, a retrospective cohort study, Meshaal [/bib_ref] The SARS-CoV-2 virus is a novel coronavirus caused by respiratory tract infection with asymptomatic to severe infection presentation. The most common simultaneity disease found in COVID-19 patients is cardiovascular disease. [bib_ref] Catastrophic Candida prosthetic valve endocarditis and COVID-19 comorbidity: a rare case, Davoodi [/bib_ref] [bib_ref] COVID-19 concomitant with infective endocarditis: a case report and review of management, Amir [/bib_ref] Since the onset of the COVID-19 pandemic, multiple reports of invasive pulmonary aspergillosis have emerged, 6 but COVID-19-associated Aspergillus endocarditis is rare. Herein, we report a rare case of native mitral valve AE in a patient with post-COVID-19-associated invasive pulmonary aspergillosis. ## | c a s e rep ort On May 13, 2022, a 63-year-old man, with severe weakness, lethargy, unilateral paralysis inability to stand up, and reduced force in the right upper limb, was admitted to the Mazandaran Heart Center affiliated with Mazandaran University of Medical Sciences, northern Iran. His past medical history revealed that he got COVID-19 about 9 months ago and was admitted to the hospital due to decreased oxygen saturation (SpO 2 ) and received remdesivir and methyl-prednisolone pulse therapy for 10 days and was discharged in a good condition. He was readmitted to the emergency department with the symptoms of red eyes and blurred vision 2 days later, and a CT scan and ophthalmological consultation highly suggestive of fungal infection, and started the treatment with amphotericin B. The three consecutive serum galactomannan assays showed one positive 6.1 and two negative results. However, despite the treatment, the visual acuity of his right eye decreased during hospitalization. Finally, after day 26 of hospitalization, the patient had been discharged with continuing voriconazole at home. Two weeks later, the patient's blood test results were as follows: ## | discuss ion Severe inflammatory responses, which are in the form of cytokine storms, are seen in patients with COVID-19. [bib_ref] Infective endocarditis and COVID 19: a systematic review, Kariyanna [/bib_ref] We can assume that increased inflammation during this COVID-19 disease, damages the heart valves and creates a suitable surface, thus binding fungi or other pathogens to the heart valve and causing infective endocarditis. Over the past year, with the increasing number of patients with COVID-19, cases of COVID-19 patients presenting with infective endocarditis have increased. [bib_ref] Infective endocarditis and Covid-19 coinfection: an updated review, George [/bib_ref] A study in 2021 suggested that the SARS-CoV-2 virus pandemic has outlined a new problem for the diagnosis of cardiovascular diseases and further care and treatment. [bib_ref] Catastrophic Candida prosthetic valve endocarditis and COVID-19 comorbidity: a rare case, Davoodi [/bib_ref] A review of endocarditis after COVID-19 showed that the highest percentage of infective endocarditis was related to the mitral valve (33%) of the heart, [bib_ref] Infective endocarditis and COVID 19: a systematic review, Kariyanna [/bib_ref] which is similar to our finding. Patients, who do not have surgery on an infected valve, pose a 100% risk of mortality while in patients who undergo cardiac valve replacement, 45% is reported. [bib_ref] Aspergillus tubingensis endocarditis: a case report and review of the literature, Born [/bib_ref] Despite appropriate treatment measures, infective endocarditis has a high morbidity and mortality rate. Mortality rates can be approximately 25%. [bib_ref] Infective endocarditis in adults: diagnosis, antimicrobial therapy, and management of complications: a..., Baddour [/bib_ref] Our report addressed the criteria of probable invasive pulmonary aspergillosis presented with a bilateral ground-glass opacity, cavitation, and bronchiectasis 9 months before the diagnosis of AE. The immunocompromising condition was steroid therapy, direct damage of respiratory virus to airway epithelium, and lymphopenia in the context of ARDS associated with COVID-19. In general, Aspergillus fumigatus has been reported to be the most common ## Ack n owled g m ents Informed consent for publication was given by the patient's spouse after the decease. ## Fu n d i n g i n fo r m ati o n The author would like to thank the Invasive Fungi Research Center of Mazandaran University of Medical Sciences, Sari, Iran for financial support (grant no. 1327). ## Co n fli c t o f i nte r e s t None. ## Data ava i l a b i l i t y s tat e m e n t

Vitamin E Acetate as a Plausible Cause of Acute Vaping-related Illness # Introduction and background "Vaping" (i.e., heating) is the practice of inhaling an aerosol created by heating a liquid or wax containing substances, such as nicotine, cannabinoids such as tetrahydrocannabinol (THC), cannabidiol, additives, and flavorings such as propylene glycol, glycerol and flavored nicotine. Vaping product use associated lung injury (EVALI, also called vaping associated pulmonary injury [VAPI]) is an acute or subacute respiratory illness with damage to the alveoli that can be severe and life-threatening. There are many available devices to generate this aerosol, including battery-operated electronic cigarettes, e-cigarettes, vape pens, or vape mods. While the specific cause of vaping-related illnesses has been widely debated among the general public and physicians, new research points to vitamin E acetate as a potential culprit in the vape-related lung illness outbreak. Vitamin E acetate is an oily chemical commonly added to THC vaping liquids to dilute or thicken them; the substance has been acknowledged as a potential toxin of concern by the Center for Disease Control and Prevention (CDC), due to its ability to remain in the lungs for long periods of time, and therefore cause complications in the lungs. The identification of vitamin E acetate as a harmful chemical in vaping liquid allows physicians to implement more concentrated care when treating vape-related illnesses and allows for the general public to gain a better understanding of the harmful substances in vaping products. Electronic cigarettes were first developed in China in the early 2000s and introduced to the US market in 2007. In the US, the product experienced explosive growth, with the number of electronic cigarette users doubling every year between 2008 and 2012. While traditional cigarettes are smoked through combustion, e-cigarettes are "vaped," and the resultant aerosols potentially contain a reduced number of potentially toxic chemicals, such as nicotine and flavorings such as diacetyl and cinnamaldehyde, as well as byproducts such as formaldehyde and acrolein caused by the potential overheating of propylene glycol, and glycerin. The Tobacco Control Act stipulates that the Food and Drug Administration shall have the authority to establish product standards for the nicotine content of combusted tobacco products. Vaping was initially marketed as a smoking cessation aid to help with cessation of cigarette smoking. E-cigarettes first took public attention in the mass media for unexpectedly blowing up, causing burns and severe facial damage. In the past decade, a number of alternative vaping products have rapidly gained consumer demand, especially in, adolescents, due to the belief that they are much safer (lower nicotine content) than traditional cigarettes, choice of advertisements different flavors and ease of access to electronic nicotine delivery systems (such as e-cigarettes and vape pens). Except for menthol, the use of flavor additives has been banned from traditional cigarettes, whereas e-cigarettes are marketed in over 7,000 different flavors. Many of those flavors are found in candy and popular soft drinks and, because adolescents are familiar with such flavors, e-cigarettes are appealing to them. Tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose-dependent manner. Data from 5,400 smokers and 2,025 former smokers have found that the average number of cigarettes smoked per day by people who regularly used e-cigarettes fell by 4.4 over about two years, compared with only 2.7 for those who did not use e-cigarettes. Sixty-seven percent more e-cigarette users than non-users quit smoking altogether. However, there were 70% more relapses among former smokers who used e-cigarettes than among those who did not use the devices. One of the most commonly known/used vape products by adolescents is the JUUL, which has twice the amount of nicotine concentrate as other e-cigarette brands, according to the American Academy of Family Physicians. The nicotine level in one JUUL pod is equivalent to that of one pack of cigarettes, and the pod consists of a mixture of nicotine salts, glycerol, propylene glycol, benzoic acid, and various flavorings. Juul Labs, Inc. is an American electronic cigarette company that became the most popular e-cigarette in the United States at the end of 2017 with a market share of 72% as of September 2018. The three main reasons e-cigarettes are attractive to young people are the belief that vaping is less harmful than smoking, and they have a lower per-user cost than traditional cigarettes and flavorings (i.e., watermelon or apple pie). Although vaping has become an integral part of the adolescent culture in recent years, the medical effects of vaping have only recently been brought to light. Many adolescents, especially, have experienced lung illnesses related to vaping, and there have been a number of people that have died due to complications from vaping-related illnesses. As of the last week of October 2019, the CDC reported that there were 1,888 cases of vape-related illnesses and 37 deaths. The death toll has risen since then, and U.S. health officials have confirmed that there have been at least 39 deaths that can be attributed to vaping illnesses, along with 2,051 of EVALI/VAPI cases. Clinicians have been instructed to remain suspicious of influenza and other respiratory infections in all patients who are experiencing respiratory symptoms that have a history of e-cigarette or vape use. Physicians have been treating vaping-related illnesses keeping in mind that each vape-related injury may be attributed to lung injuries, infections, or both. The possible causes for these complications are still widely debated, as many believe these illnesses could be caused by a multitude of ingredients found in vape. A case study of hard-metal pneumoconiosis, published in the European Respiratory Journal, researchers tested the patient's e-cigarette, which used with cannabis, found cobalt in the vapor was released, including other toxic metals-nickel, aluminum, manganese, lead, and chromium. Metal-induced toxicity in the lung can result in long-term, if not, permanent scarring of the lungs. ## Review The ingredients of vape that are suspected of contributing to the development of vapingrelated illnesses are THC and vitamin E acetate. THC is an ingredient used in many vape products, and many patients experiencing vape-related complications have admitted to using THC-containing products in the past, leading the FDA to believe that THC may play a role in the vape-related illness outbreak. The FDA has issued a public warning to stop using THCcontaining vape products, as the compound may be contributing to lung illnesses related to vaping. Specifically, vitamin E acetate is most commonly used as an additive in THC-containing vape/e-cigarette products; vitamin E acetate is an oily chemical added to THC vaping liquids used to thicken or dilute them. A vape-related injury concerning a teenage boy in Canada has recently gained the media's attention as well. The 17-year-old boy vaped "intensively," adding THC to his devices. He initially showed symptoms aligning with bronchiolitis (lung condition normally caused by a bacterial or viral infection), but many patients that have vape-related illnesses in the United States have experienced damage to the alveoli; this type of injury was not found. Instead, his case aligned more with an injury called "popcorn lung," an ailment most commonly seen in factory workers of microwave popcorn plants nearly 20 years ago. This new vape-related case calls for further exploration into the toxicity of vape liquid, as the patient's condition could have been caused by the THC added to the vaping devices, or the chemical that affected factory workers in the past -diacetyl. Diacetyl is present in many e-cigarette flavors. The American Lung Association has called for the FDA to require that diacetyl and other hazardous chemicals be removed from e-cigarette cartridges. While it is still widely debated which particular component of vape liquid is the cause of illness, vitamin E acetate, specifically, has been identified as a potential culprit in vape-related illnesses. The New York Times recently reported an analysis of lung fluid samples from 29 patients with vaping-related illnesses (including two who died), and the analysis suggests that vitamin E acetate is a "very strong culprit" in causing lung injuries. The lung fluid samples were collected from patients across the United States so that these findings may have implications nationwide. Moreover, Dr. Anne Schuchat, principal deputy director of the CDC, explained, "For the first time, we have detected a potential toxin of concern, vitamin E acetate, from biological samples from patients… The analysis provided evidence of vitamin E acetate at the primary site of injury in the lungs". Vitamin E acetate is sticky, giving it the ability to remain in the lungs. THC was also reported to be found in 82% of samples from 28 patients, which was remarkable as THC tends to leave the lungs quickly. The evidence on how vitamin E acetate affects the lungs of vape users is notable because vitamin E acetate has been acknowledged as a majorly harmful chemical that may be contributing to vape-related illnesses and deaths. Lung scans have revealed different outlines of lung parenchyma suggesting possible different processes in injury. One pattern points to lipoid pneumonia which can occur with lipid containing ingredients or oils aerosolized into the airways causing inflammation and compromised function. The respiratory epithelium has a complicated network of extracellular membranes essential for breathing and survival. Surfactant membranes form a stable monolayer at the air-liquid interface, reducing the surface tension at the air-liquid interface, therefore stabilizing the lung against collapse and helping lungs expand. Oil in the lung interferes with this ordered/disordered lipid phase coexistence in lung surfactant with alterations in phase coexistence. The American Medical Association has made calls for a ban on vaping products, and Washington state has now banned vape products containing vitamin E acetate, thought to be linked to illness. Although the substance is not banned in the United States and has not been officially declared as a deadly substance, many states are making advances to ban the use of the chemical in vape products. States like Massachusetts are considering a ban on flavored tobacco and vape products, and in New York, Manhattan is expected to become the largest city to ban all vaping flavors except tobacco. Other states that have already banned the use of vitamin E acetate in vape products include Colorado and Ohio. Greater public awareness of this deadly condition helps with implementing comprehensive, population-based interventions for this preventable disease. As of November 13, 2019, there were 2,172 confirmed and probable lung injury cases "associated with the use of e-cigarette or vaping, products as reported by 49 states (all except Alaska), the District of Columbia, Puerto Rico, and the U.S. Virgin Islands as reported by the CDC. 42 deaths have been confirmed in 24 states and the District of Columbia: Alabama, California (4), Connecticut, Delaware, the District of Columbia, Florida, Georgia (3), Illinois (4), Indiana (4), Kansas (2), Massachusetts (2), Michigan, Minnesota (3), Mississippi, Missouri, Montana, Nebraska, New Jersey, New York, Oregon (2), Pennsylvania, Tennessee (2), Texas, Utah, and Virginia". Vaping, may also have harmful psychological effects with a strong association between vaping, major depression and suicidal behavior as reported in a large new study. The likely contributing culprit, nicotine. Prevalence of lung disease attributable to vaping is likely under reported as cases brought to the CDC are some of the most severe. For now EVALI remains a diagnosis that is made after exclusion of other conditions and needs to be reported to the CDC. # Conclusions Tobacco use continues to devastate public health. Given the popularity of vaping and ecigarettes, urgent public health call to action to this condition is needed, especially in light of the EVALI/VAPI death complications. Identification of vitamin E acetate and other possible hazardous chemical additives such as diacetyl as the contributors in the vape-related lung illness outbreak is critical to both physician and public understanding of the dangers of vaping. Identifying the particular substances in a vape that can cause lung illness and/or complications from vaping can be the crucial step toward identifying and eliminating this fatal condition, which continues to lack formal diagnostic criteria and standardized therapy. Teenagers are particularly vulnerable to the addictive flavors to nicotine. Given that many e-cig flavors, many found in candy and popular soft drinks, the addition of flavorings are likely preying on ecigarette experimentation in young people. # Additional information disclosures ## Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Boudi et al. Cureus 11(12): e6350. DOI 10.7759/cureus.6350 2 of 5 Boudi et al. Cureus 11(12): e6350. DOI 10.7759/cureus.6350 3 of 5 Boudi et al. Cureus 11(12): e6350. DOI 10.7759/cureus.6350 5 of 5

Random selection of factors preserves the correlation structure in a linear factor model to a high degree In a very high-dimensional vector space, two randomly-chosen vectors are almost orthogonal with high probability. Starting from this observation, we develop a statistical factor model, the random factor model, in which factors are chosen stochastically based on the random projection method. Randomness of factors has the consequence that correlation and covariance matrices are well preserved in a linear factor representation. It also enables derivation of probabilistic bounds for the accuracy of the random factor representation of time-series, their cross-correlations and covariances. As an application, we analyze reproduction of time-series and their cross-correlation coefficients in the well-diversified Russell 3,000 equity index.OPEN ACCESSCitation: Tanskanen AJ, Lukkarinen J, Vatanen K (2018) Random selection of factors preserves the correlation structure in a linear factor model to a high degree. PLoS ONE 13(12): e0206551. https:// # Introduction ## Vectors in a high-dimensional space In a high-dimensional vector space, any two unit-length random vectors with random independent components are typically nearly orthogonal with respect to each other [bib_ref] Dimensionality reduction by random mapping: Fast similarity computation for clustering, Kaski [/bib_ref]. To have a concrete example, one could consider random vectors in R d whose components are taken independently from a common distribution with a zero mean and a variance 1/d. Then it is straightforward to check that the expectation of the square of the length of a vector is one and the expectation of a scalar product between any two independently chosen vectors is zero. Mathematical probability theory has techniques which allow deriving explicit estimates for correlations of such high dimensional random vectors and exploring their dependence on the random distribution. Detailed examples will be given later and further discussion may be found in S1 Appendix. For example, it is possible to check whether the expected length squared and scalar products in the above random vector example are reached with a high probability. If the common distribution is Gaussian, we may conclude from the results given in Section 2.4.3 that the variances of both of observables are O(1/d) as d ! 1. By Chebyshev's inequality, these estimates imply that the respective means are reached up to some fixed accuracy with a probabilityPlease see https://github.com/ajtanskanen/Random-selection-of-factors-demo for a numerical demonstration. The maximal number of nearly orthogonal unit vectors is closely related to generic geometric properties of high-dimensional spaces. For instance, even though the maximal number of exactly orthogonal unit vectors from R d is d, the maximal number of nearly orthogonal unit vectors grows exponentially fast with d. More precisely, suppose we call two unit vectors εquasiorthogonal if the absolute value of their inner product is at most ε for some ε > 0. Then the maximal size of a collection of ε-quasiorthogonal vectors in R d grows at least exponentially fast in d for any fixed ε > 0, as proven in [bib_ref] Quasiorthogonal dimension of Euclidean spaces, Kainen [/bib_ref]. These observations have relevance to time-series analysis, since a long time-series corresponds to a vector in a high-dimensional vector space and orthogonality of vectors corresponds to uncorrelatedness of time-series. Time-series corresponding to two randomly selected vectors are therefore expected to be in general almost uncorrelated. When the length of the time-series increases, or equivalently, dimension of the corresponding vector space increases, the probability that two randomly-selected time-series are uncorrelated increases. If we select a set of, say, n vectors randomly, these vectors are approximately orthogonal to each other if the dimension of the space is sufficiently high. Here, high-dimensionality of the data may even be an asset: in a high-dimensional space, almost any set of random vectors yields an almost uncorrelated set of factor time-series that can be used as a basis for a linear factor model. ## Factor models Factor models are extensively used in financial applications to model asset returnsand to decompose them to loadings of risk factors. Factor models have provided insight into, e.g., the drivers of asset returns, and they have influenced the way risks are managed. In short, factor modeling can be considered a huge success. The three main types of factor models are macroeconomic factor models, fundamental factor models and statistical factor models [bib_ref] The three types of factor models: A comparison of their explanatory power, Connor [/bib_ref]. In a macroeconomic factor model, the factors are defined via economic theory and they are observed in addition to, and externally to, the security returns data. In a fundamental factor model, the aim is to find observable asset characteristics, e.g. financial ratios, capable of explaining the behavior of market stock prices, that are often extrinsic to the asset time-series. The explanatory fundamental and economic variables can be highly correlated with each other, which may cause multicollinearity of factors. Returns predicted by a fundamental factor model may then be more correlated than the observed returns, which is the main reason for the inclusion of specific risk components in a factor model. Classical factor models include only a handful of factors. One of the best-known factor model in the literature is the Capital Asset Pricing Model (CAPM), which assumes that a single risk factor, the market, drives returns in a portfolio of assets. Recent increase in computational power has enabled development of models with a large set of factors. A number of factor models have extended this view [bib_ref] The arbitrage theory of capital asset pricing, Ross [/bib_ref] [bib_ref] Common risk factors in the returns on stocks and bonds, Fama [/bib_ref]. Today, factor models are popular in market risk modeling, e.g., the Barra modelsdepend on hand-picked market factors to explain behavior of the analyzed financial instruments. Statistical factor models are a commonly-used alternative for fundamental and macroeconomic factor models. In a statistical factor model, factors are extracted from asset returns, and the set of factors may be large. The principal component analysis (PCA) is an example of a statistical technique for finding factors from asset time-series. PCA works well when the analyzed time-series are highly correlated, which may indicate the presence of a common driver. Applications of PCA include models of interest rate term structure, credit spreads, futures, and volatility forwards. It is often the case that several principal components have an intuitive financial interpretation. In ordered highly-correlated systems, the first principal component captures an almost parallel shift in all variables and is generally labeled the common trend component. The second principal component captures an almost linear tilt in the variables, while the higher order principal components capture changes that are quadratic, cubic and so forth. In the equity markets, the higher order principal components may often, but certainly not always, be interpreted as market movements caused by different investment style tilts. Principal components found using PCA provide consistent estimators of the true latent factors in the limit of both time and cross-sectional size approaching infinity [bib_ref] Forecasting using principal components from a large number of predictors, Stock [/bib_ref]. It can be shown that this extends consistent estimation of the classical factor model with non-correlated errors to approximate factor models with cross-correlated and sectionally-correlated error terms [bib_ref] Forecasting using principal components from a large number of predictors, Stock [/bib_ref]. ## Choice of factors The choice of factors clearly influences the ability of a factor model to explain investment risk of a portfolio, in particular when the factor model consists of only a few carefully-chosen factors. If the number of factors is large compared to the number of time-series analyzed, it may not much matter which factors are chosen as long as the factors span a sufficiently large sample space. Even then, relative importance of factors is of interest in risk management. There are situations in which the use of a larger number of time-series may actually result a worse factor estimate than a smaller number of time-series [bib_ref] Are more data always better for factor analysis?, Boivin [/bib_ref]. A significant amount of recent literature has been devoted to addressing the issue of consistent estimation under conditions where the number of time-series is large compared to the length of time-series [bib_ref] Cleaning large correlation matrices: tools from random matrix theory, Bun [/bib_ref] [bib_ref] Nonlinear shrinkage estimation of large-dimensional covariance matrices, Ledoit [/bib_ref]. Now, imagine that one is given a set of factors that are in no way fitted to the problem in question. Would this, rather arbitrary set of factors enable description, or better yet, reduction of the data to a smaller set of factors? It is not clear how well an arbitrary set of factors would enable analysis-or at least description-of the risk. This is the issue that we analyze in this study: take a random set of factors and see whether it enables reproduction of the data and its interdependencies. ## Correlation structure and random matrices Analysis of risk in an investment portfolio requires that risks of individual instruments are combined into the risk of the portfolio. This can be accomplished using dependence structures, e.g., correlation matrix. It is no coincidence that dependence structures of financial time-series are central in modern investment theory. The recent explosion of available data has brought new issues with time-series analysis [bib_ref] Cleaning large correlation matrices: tools from random matrix theory, Bun [/bib_ref]. To get around these issues, new methods of analysis are needed. One such tool is the random matrix theory [bib_ref] Random matrix theory, Edelman [/bib_ref] , which can be used to alleviate the issues of "Big data" [bib_ref] Cleaning large correlation matrices: tools from random matrix theory, Bun [/bib_ref]. For example, there are issues of correlation matrix estimation when there are more time-series than data points in each time-series, and the random matrix theory can be used to improve empirical estimates of correlation matrices [bib_ref] Cleaning large correlation matrices: tools from random matrix theory, Bun [/bib_ref]. In the random matrix theory, things often get less complex when dimension of the problem increases. Another important feature of random matrix theory is universality. Most of the eigenvalue spectrum of correlation matrix for the Standard & Poor's 500 Index (S&P 500) equities is part of the continuous eigenvalue bulk, only about 20 eigenvalues of 500 total are not. The bulk of the power spectrum of the S&P 500 returns is very similar to that produced by Gaussian Orthogonal Ensemble, in which the asymptotic eigenvalue distribution is given by the Marchenko-Pastur law. For this reason, it has been argued that eigenvalues in the bulk do not carry information, however, this view has recently been challenged [bib_ref] Fine structure of spectral properties for random correlation matrices: An application to..., Livan [/bib_ref]. Most eigenvalues in the spectrum of the cross-correlation matrix of stock price changes agree surprisingly well with universal predictions of random matrix theory [bib_ref] Universal and nonuniversal properties of cross correlations in financial time series, Plerou [/bib_ref]. This enables construction of a model with a spectrum that exhibits the features found in S&P 500 spectrum: a few large eigenvalues and a bulk part [bib_ref] Collective origin of the coexistence of apparent random matrix theory noise and..., Malevergne [/bib_ref]. The 20 eigenvalues not part of the bulk may correspond to the market movements and sectors, but most of the eigenvalue spectrum does not appear to correspond to linear factors. For a more detailed discussion of eigenvalues in factor models, see [bib_ref] Fine structure of spectral properties for random correlation matrices: An application to..., Livan [/bib_ref]. There also is the chicken-and-egg problem associated with factors: factors exist because stocks are correlated; stocks are correlated because of a common factor impacting them [bib_ref] Collective origin of the coexistence of apparent random matrix theory noise and..., Malevergne [/bib_ref]. The apparent presence of factors is a consequence of the collective, bottom-up effect of the underlying time-series [bib_ref] Collective origin of the coexistence of apparent random matrix theory noise and..., Malevergne [/bib_ref]. A naive use of the sample covariance matrix has drawbacks [bib_ref] Honey, I shrunk the sample covariance matrix, Ledoit [/bib_ref]. Shrinkage of the sample covariance matrix toward a structured or a constant matrix can be used to reduce extremal estimation errors [bib_ref] Honey, I shrunk the sample covariance matrix, Ledoit [/bib_ref]. Marchenko-Pastur law can be used to improve empirical estimates of correlation matrices further [bib_ref] Nonlinear shrinkage estimation of large-dimensional covariance matrices, Ledoit [/bib_ref]. Given the above examples, it is clear that the random matrix theory can be used for analyzing financial time-series. We make use of random matrices to develop a factor model and to analyze the properties of correlation matrix preservation in a generic linear factor model. ## The random projection A good starting point for our analysis, and for developing a random factor model is the random projection method [bib_ref] Random projection in dimensionality reduction: applications to image and text data, Bingham [/bib_ref]. The random projection allows one to reduce dimensionality of the investigated problem, often substantially, while preserving the structure of the problem. The random projection consists of a projection of data to a lower-dimensional space by a random matrix. The random projection method has been used, e.g., to reduce the complexity of the data for classification purposes [bib_ref] Self organization of a massive document collection, Kohonen [/bib_ref] , for structure-preserving perturbation of confidential data in scientific applications [bib_ref] Random projection-based multiplicative data perturbation for privacy preserving distributed data mining, Liu [/bib_ref] , for data compression [bib_ref] Random projection in dimensionality reduction: applications to image and text data, Bingham [/bib_ref] , for compression of images, and in the design of approximation algorithms [bib_ref] Random projection, margins, kernels, and feature-selection, Blum [/bib_ref]. The previous literature has compared the performance of the random projection method with PCA, and found situations in which the random projection method performs comparably or even better than PCA. In the compression of image data and in text clustering, random projection method performed significantly better than PCA [bib_ref] Random projection in dimensionality reduction: applications to image and text data, Bingham [/bib_ref]. Random projection compares favorably with PCA, although PCA is more accurate with small number of dimensions [bib_ref] Face recognition experiments with random projection, Goal [/bib_ref]. In text clustering, a PCA-based method provides better accuracy with small number of dimensions, while with high number of dimensions the random projection method dominated [bib_ref] Comparing and combining dimension reduction techniques for efficient text clustering, Tang [/bib_ref]. In the analysis of five image data sets and five micro array data sets, PCA dominated with a small number of dimensions but its performance deteriorated when the dimensions of the data increased, while random projection dominates at high number of dimensions (cross-over occurs at 15-150 dimensions depending on the data set) [bib_ref] Reducing high-dimensional data by principal component analysis vs. random projection for nearest..., Deegalla [/bib_ref]. The above results differ from the factor model setting in important ways, and the results cannot directly be applied to factor modeling. Still, they suggest that the random projection-based methods may be usable instead of PCA also in factor modeling. ## This study It is largely an open question whether and how well randomly-chosen factors can be used in a linear factor model to describe large data sets. This is the issue we approach in this study. For this purpose, we develop a factor model based on randomly-chosen factor time-series, the random factor model. We show that randomness of factors has certain desirable properties, such as well-defined probabilistic limits on the accuracy of the factor representation. In addition, randomly-chosen factors are almost orthogonal with high probability, and with a proper normalization, they are expected to be orthonormal. We also show how the random factor model converges toward the modeled data when the number of factors increases, and that a random factor model preserves pair-wise correlations well with high probability. The article is structured as follows. In Section 2, we develop the random factor model based on the random projection method and derive theoretical results describing the model (more details can be found in S1 Appendix). For example, we show that randomness of factors enables derivation of theoretical results on the accuracy of the model. From the econometric point of view, a random factor modeling can be viewed as a projection of time-series to a collection of factors that are almost non-autocorrelated and non-crosscorrelated, as will be seen in Section 2. As an application of the random factor model, Section 3 provides an analysis of the correlation matrix of the Russell 3,000 equity index using the factor models described in Section 2. We analyze the ability of random factor models to reproduce equity log-return time-series and their correlations and covariances. The reproduction of data in a random factor model is compared with a reproduction obtained using principal component analysis both at the individual time-series level and at the dependence structure level. In Section 4, we compare different random factor models and show that the results, or rather their accuracy, are quite universal. In Section 5, we discuss the results and their possible implications. In S1 Appendix, we prove an extended version of the Johnson-Lindenstrauss theorem appropriate for the random factor model. It gives probabilistic bounds on the accuracy of the correlation and covariance preservation in a random factor model. ## Random factor model ## Notations Let d observations of time-series Z : N ! R be viewed as a vector in d-dimensional space R d , where each observation of the time-series corresponds to one coordinate of the vector. The set of N such time-series can be packed into matrix X 2 R d�N , in which observations are in columns. We assume that the time-series data has been preprocessed, so that each time-series is averaged to zero, that is, P m X mb = 0 for each b = 1,. . ., N. We employ sample statistics in this study. Definitions for mean μ, variance σ 2 and covariance C are [formula] m x ¼ 1 d X d m¼1 x m ; C x;y ¼ 1 d À 1 X d m¼1 ðx m À m x Þðy m À m y Þ; s 2 x ¼ C x;x ;ð1Þ [/formula] where x; y 2 R d . The central parameters used in this study are summarized in [fig_ref] Table 1: The central parameters used [/fig_ref]. ## Linear factor models A linear factor model describes the target data set as a loading-weighted sum of factors (e.g.,. Let F ¼ ½F 1 F 2 ::: F k � 2 R d�k contain d observations of the factors j = 1, 2,. . ., k, F j 2 R d�1 . [formula] Then time-series X b 2 R d�1 , where X b is b:th column of matrix X, b = 1,. . ., N, [/formula] can be represented as a sum of products of factor loadings L bj 2 R and factors F j , that is, [formula] X b ¼ X k j¼1 L bj F j þ � b ;ð2Þ [/formula] where � b is an idiosyncratic risk component. Since we collect the observations into columns of X and F, the formula (2) is written in a matrix form as X = FL T + �. Factors in F may or may not be directly observable in the market data. For observed factor time-series, it suffices to project the data to factors to get loadings. For unobserved factor timeseries, method such as PCA or another optimization method, e.g., Maximum Likelihood Estimation, is required to find the factors and their loadings. ## Random projection Random factors are here chosen using the random projection method. The key idea of random projection is based on the Johnson-Lindenstrauss lemma [bib_ref] Random projection in dimensionality reduction: applications to image and text data, Bingham [/bib_ref] [bib_ref] Extensions of Lipschitz mappings into a Hilbert space, Johnson [/bib_ref] : if points in a high-dimensional space are projected onto a randomly selected subspace of suitably high dimension, then the distances between the points are approximately preserved. A suitably high-dimensional subspace has dimension proportional to log(N)/ε, where N is the number of time-series and ε the desired accuracy [bib_ref] An elementary proof of a theorem of Johnson and Lindenstrauss, Dasgupta [/bib_ref]. Random projection Q : [formula] R d�N ! R k�N of matrix D 2 R d�N is a mapping defined by Q(D) = BD, where k; d; N 2 N. Here matrix B is realization B(ω) of a random variable-valued k × d matrix. [/formula] From this point on, we will not differentiate between random variables and their realizations and tacitly assume that the distinction can be inferred from the context. A large variety of probability distributions can be used to construct projection matrix B (more on this in Section 3.3). The most obvious choice is to assume that matrix B is taken from the matrix-variate normal distribution with independent entries, that is, from N k×d (0, 1 k × 1 d ). Then each element is N(0, 1)-distributed and independent of other elements. ## Random factor model 2.4.1 definition and properties. We define the random factor model (RFM) for data set X 2 R d�N via a projection. Strictly speaking, the matrix P is not a projection matrix since it typically does not satisfy the equality P 2 = P. However, since its range is a lower dimensional subspace, we use the term "projection" also to describe P, in analogy with the definition of the term "random projection" in Section 2.3. P : [formula] R d�N ! R d�N , PX ¼ aB T BX;ð3Þ [/formula] where B 2 N k×d (0, 1 k × 1 d ) is a k × d-dimensional random variable, elements of which are independent and normally distributed, and a > 0 is a normalization constant. Mapping (3) can be interpreted as a linear factor model by setting [formula] L ¼ a a 0 X T B T ; ð4Þ F ¼ a 0 B T ;ð5Þ [/formula] where a 0 > 0 is a constant related to factor normalization, as discussed in Section 2.4.3. Then F 2 R d�k behaves as a matrix of k d-dimensional factor time-series. It is worth stressing that matrix F consists of random time-series that in no way depend on the data. L 2 R N�k is a matrix of k factor loadings for the N time-series. Projection P can be factored as [formula] PX ¼ FL T :ð6Þ [/formula] Defining � � = X − PX yields an approximate factorization [formula] X ¼ FL T þ � �ð7Þ [/formula] for data matrix X. We will analyze Eq (7), and in particular error term � � , further in the following. Eq (7) shows that data matrix X can be approximately decomposed into a product of two components. As an aside, let us mention that we could equally well have considered a random projection in the equity direction instead of the above time-series direction. This can be accomplished using a matrix Q = aR T R, where a > 0 and R 2 R k�N is a random matrix, and then considering XQ as the projected matrix. This naturally leads to a factor model interpretation with a loading matrix a a 0 R T and a factor matrix a 0 XR T . In analogy to the earlier terminology, this model could be called random loading model. The properties proven later for the random projection P then immediately carry over to the projection Q, one merely needs to replace "d" with "N" in all of the results. However, from the point of view of the time series, the two projection methods PX and XQ could behave differently. For instance, if there are more pronounced correlations between different equities at a fixed time than between the same equity at two different times, then one would expect to need larger values of k in the projection XQ than in the projection PX to reach the same level of accuracy in the approximation. It is also possible to apply both random projections simultaneously and study PXQ instead of PX or XQ. This double-sided projection would still have properties very similar to the one-sided projections, as long as the random matrices B and R are chosen independently of each other. Since the three alternatives are on a technical level very similar, we focus only on the choice PX in the following. As the next step, we need to find a suitable constant a so that standard deviation, covariance and the expected value of the data are preserved, if possible. Under these conditions, � � should be close to zero. Different choices of a yield slightly different properties for the RFM, but it turns out that we cannot satisfy all these requirements at the same time if we base matrix B on the normal distribution. Here we concentrate on preserving the covariance matrix C x,y in the projection. Then normalization constant a > 0 must be such that expectation with respect to N k×d (0, 1 k × 1 d ) is preserved, that is, [formula] E½C Px;Py � ¼ C x;yð8Þ [/formula] for any zero-mean vectors x; y 2 R d�1 . It is worth stressing that the expectation in Eq (8) is taken over random factor models, not over time-series x and y. Theorem 1.1 in S1 Appendix shows that this is possible but only if we choose a ¼ 1= ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi kðk þ dÞ p . Let a have this value from this point on. The expected covariance between time-series x and y is then preserved, regardless of the number of factors used. Since E½s 2 [formula] Px � ¼ E½C Px;Px � ¼ C x;x ¼ s 2 x , [/formula] our choice of a also preserves time-series variance. This result shows that an RFM is expected to fulfill the consistency requirement of variance, that is, it shows that [formula] lim d!1 E½s 2 Px;d � ¼ lim d!1 s 2 x;d ¼ s 2 x;pop ;ð9Þ [/formula] where s 2 x;pop is the population variance and s 2 x;d is the sample variance in dimension d. An application of the Jensen's inequality implies that E½s Px � � s x , that is, volatility is not overestimated. Representation (7) always preserves the average of a zero-mean vector The RFM is expected to reproduce mean, variance and covariance of time-series x. Component-wisely, the random factor model is expected to converge to the observed component values in the limit of large number of factors. (8) does not state that each RFM always preserves the covariance matrix. Nevertheless, it is reasonable to assume that an RFM approximately preserves the covariance matrix. Next we will analyze how well an RFM will typically preserve the covariance matrix. [formula] x 2 R d , that is, E½m Px � ¼ 0. In contrast, [/formula] ## Covariance preservation. eq But first, it is worth recalling that Johnson-Lindenstrauss theorem [bib_ref] Extensions of Lipschitz mappings into a Hilbert space, Johnson [/bib_ref] [bib_ref] An elementary proof of a theorem of Johnson and Lindenstrauss, Dasgupta [/bib_ref] [bib_ref] On variants of the Johnson-Lindenstrauss lemma, Matoušek [/bib_ref] gives probabilistic bounds for the accuracy of distance preservation in the random projection. A number of versions of Johnson-Lindestrauss theorem have been proven, however, in all versions known to us, it is assumed that random variables have zero expectation. Matrix B T B 2 R d�d is a singular Wishart matrix (also known as an anti-Wishart matrix), which has non-zero expectation, d − k zero eigenvalues and k non-zero eigenvalues. Since matrix B T B has non-zero expectation, it was not a priori clear if a Johnson-Lindenstrauss type theorem holds. Theorem 1.1 proven in S1 Appendix fills this gap for the present type of anti-Wishart matrices, and it also contains a detailed derivation of the above expectation values for an arbitrary value of the scaling parameter a. We have collected in Corollary 1.2 in S1 Appendix the corresponding results for the choice which preserves the sample covariance matrices in expectation, for a ¼ 1= ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi kðk þ dÞ p . The precise control of fluctuations in the covariance estimates requires nontrivial combinatorial computations, given in S1 Appendix. As proven in the Corollary, for every b > 0 and non-random vectors u; v 2 R d , with μ u = 0 = μ v , we have [formula] P jC Pu;Pv À C u;v j � b � � � 8 kb 2 s 2 u s 2 v :ð11Þ [/formula] Inequality (11) gives bounds on the accuracy of covariance preservation for an arbitrary random factor model. Here probability is taken with respect to an ensemble of random factor models. Since our proof is based on the Chebyshev inequality, there could still be room for improvement in the estimate. Also, as noted in Remark 1.3 in S1 Appendix after the proof, the prefactor 8 in Eq (11) is not always optimal, and it could be reduced to 2 in the regime d � k. Hence, if s 2 u ; s 2 v � 1, the probability that covariance of vectors u and v is preserved in a random factor model more accurately than bound b is at least 1 − 8/(kb 2 ), where k is the number of factors. In general, we can set b = εσ u σ v , with ε > 0, and also conclude that the accuracy, relative to the sample variance scale σ u σ v , is at least ε with a probability of at least 1 − 8/(kε 2 ). For the bound to be informative, it is necessary that ε > k −1/2 . The error in the covariance estimate decreases at least inversely with the number of factors in almost any random factor model. Given a sufficient number of factors, covariance of any two time-series can be approximated with an arbitrarily high accuracy using an RFM. This and the fact that random factors are in no way fitted to the data suggest that the typical accuracy of an RFM depends mainly on the number of factors k. Corollary 1.2 in S1 Appendix also gives a bound on how accurately correlation between projected vectors is preserved. Correlation Corr(u, v) coincides with covariance C u,v when σ u = σ v = 1. Then inequalitygives a lower bound on how well C Pu,Pv approximates correlation between u and v. These results can be summarized as a statement about the projected matrix PX as follows: [formula] P 1 s X b s X c jC ðPXÞ b ;ðPXÞ c À C X b ;X c j < ε " # � 1 À 8 kε 2 ;ð12Þ [/formula] valid for any b, c = 1, 2,. . ., N and ε > 0. ## Almost orthogonality. Orthogonality is a desirable property of a factor set. An orthogonalization procedure can be used to obtain an orthogonal factor set, but orthogonalization is computationally expensive. Fortunately, orthogonalization is not a necessary step in the RFM. Given any two random factors (as defined above), their inner product is expected to be orthogonal, that is, [formula] E X d m¼1 F mj F mj 0 " # ¼ ða 0 Þ 2 X m E½B j 0 m B jm � ¼ ða 0 Þ 2 dd j 0 ;j :ð13Þ [/formula] This shows that with the choice a 0 ¼ 1= ffi ffi ffi d p , the factors F j 0 and F j are expected to be orthonormal as a consequence of the properties of normally distributed random variables. Hence, any two random factors are non-collinear with respect to each other. Using Theorem 1.1 in S1 Appendix we can also compute the variance of the inner product, [formula] Var X d m¼1 F mj F mj 0 " # ¼ 1 d ðd j 0 ;j þ 1Þ � 2 d :ð14Þ [/formula] Higher cumulants approach zero even more rapidly, as can be seen by analyzing the cumulant generating function [formula] ln E e l P d m¼1 F mj F mj 0 � � ¼ ( À d 2 ln 1 À 2 l d À � ; when j ¼ j 0 ; À d 2 ln 1 À l 2 d 2 À � ; otherwise;ð15Þ [/formula] and its series expansion in λ. When j = j 0 , nth cumulant is of order O(d 1−n ). When j 6 ¼ j 0 , cumulants are of order O(d 1−n ) for even n and zero otherwise. Convergence to the Normal distribution in the limit of large d then follows by the standard arguments (e.g., [bib_ref] Probability and Measure, ser, Billingsley [/bib_ref] When d is large (�1, 000), standard deviation is only a fraction of the expectation for diagonal elements. Fluctuations around zero are small for non-diagonal elements. The cumulant expansion shows that the factors are almost orthonormal even at a relatively low dimension. In addition, the factors are on average orthogonal to the error term � � : since � � is an even polynomial of B:s and F j is linear in them, we have E½ P d m¼1 F mj � � mb � ¼ 0 for all j = 1, 2,. . ., k and b = 1, 2,. . ., N. # Principal component analysis PCA is a well-known technique which uses a linear transformation to form a simplified data set retaining the characteristics of the original data set. In investment risk measurement, PCA is used to explain the covariance structure of a set of market variables through a few linear combinations of these variables. The general objectives of using PCA are to reduce the dimensions of covariance matrices and to find the main risk factors. The risk factors can then be used to analyze, e.g., the investment risk sources of a portfolio, or to predict how the value of the portfolio will develop. Projection to principal components is most directly obtained using the singular value decomposition. Given data matrix X 2 R d�N , SVD decomposes it as X ¼ P L DP T R , where P L 2 R d�d is matrix of left singular vectors, P R 2 R N�N is matrix of right singular vectors, and D 2 R d�N is the rectangular diagonal matrix of singular values. PCA-based factor representation of X is given by X = FL T , where L ¼ P R 2 R N�N gives the factor loading matrix and F ¼ P L D 2 R d�N defines the factors of the N equities. When reducing the dimensions of the original dataset, the first k principal components with the largest eigenvalues are chosen to represent the original dataset. This yields an approximation of the data matrix using a subset of factors. A k-factor approximation of matrix X is given by F (k) (L (k) ) T , where L ðkÞ 2 R N�k contains the first k factor loadings, F ðkÞ 2 R d�k contains the components of the first k factors from P L D. It can be shown that in the mean-error sense, PCA gives the best linear k-factor approximation to matrix X (e.g., [bib_ref] Principal component analysis and optimization: A tutorial, Reris [/bib_ref] [bib_ref] The approximation of one matrix by another of lower rank, Eckart [/bib_ref]. Principal components correspond to directions along which there is most variation in the data. However, there are no guarantees that pair-wise distances are preserved in PCA. PCA yields the relative importance of the most important risk sources (defined in factor matrix F) in an investment portfolio. The relative importance of risk factors is shown by the size of eigenvalues. The eigenvectors with highest eigenvalues correspond to the most important risk factors. Loadings then tell how much investment instruments depend on these factors. Nevertheless, it should be stated that PCA aims to capture total variation, not correlations. ## Comparison of factor models Despite appearance, the RFM and PCA share many features. In both models, the data can be represented as FL T , where L contains k factor loadings and F defines k factor time-series with d observations. In PCA, the most important eigenvectors are found by choosing the largest eigenvalues. No such ordering is available for random vectors. A random vector is essentially as good as the next random vector as a factor. The RFM has an almost orthonormal factors, while PCA yields strictly orthonormal factors. After finding the factors, both the RFM and PCA project the data to these vectors. The ways in which the RFM and PCA end up with representations of the data matrix are quite different: in PCA, data is projected along principal components (factors) and only the desired set of these projections (loadings) are kept. In the RFM, the data is projected along the random factors. The main difference is in the way that factors are chosen. PCA requires O(d 2 N) + O(d 3 ) operations, while the RFM requires O(kdN) operations, given the factor time-series. Since the number of factors is typically significantly smaller than the dimension of data, the RFM is computationally much more efficient than PCA. We do not aim at proving the supremacy of the RFM over PCA. We rather use PCA as a yardstick against which the RFM is compared. It is worth remembering that there is no fitting to data in the RFM, so one could reasonably expect that PCA would surpass RFM in every respect in data experiments. ## Application to the russell 3,000 equity index The Russell 3,000 index (ticker RAY in Bloomberg) measures the performance of the 3,000 largest US companies. The index represents about 98 percent of the investable US equity market. Here, we investigate how well a random factor model reproduces log-returns of the Russell 3,000 equities and their cross-correlations, and compare the results to those obtained using PCA. For our analyses, we employ daily log-returns of Russell 3,000 equities from 2000-01-03 to 2013-02-20 (in total 3,305 observations). This interval contains several phases of the business cycle and certain special events, e.g., the crash of September 2008. Of the 3,000 constituent time-series in the index, we used a subset of 1,591 time-series with continuous daily data covering the entire period. To apply the analysis methods, the data is normalized by subtracting mean of each return time-series and by dividing by its standard deviation. As is demonstrated in [fig_ref] Fig 1: provides three examples of equity time-series reproduction using the RFM and PCA [/fig_ref] random selection of factor time-series enables reproduction of (normalized) equity time-series, when the number of factors is sufficiently high. The number of factors is not limited to the number of time-series in the RFM, since the random factors do not necessarily span the entire space in which time-series may have values. Only in the limit of large number of factors is the entire space covered. ## Reproduction of time-series Both PCA and the RFM provide good reproductions of the data [fig_ref] Fig 1: provides three examples of equity time-series reproduction using the RFM and PCA [/fig_ref] , however, there are deviations from the data in each reproduction. In the root mean square error (RMSE) sense, PCA gives a better reproduction of the time-series than the RFM [fig_ref] Fig 2: The difference is that Fig 5 shows [/fig_ref] , as one would expect. RMSE in the reproduction of the entire data set is 0.79 in PCA vs 1.37 in the RFM with 10 factors [fig_ref] Fig 1: provides three examples of equity time-series reproduction using the RFM and PCA [/fig_ref]. ## Volatility The RFM reproduces volatility of the time-series almost exactly even with a small number of factors, while in PCA volatility estimates improve pronouncedly with more factors [fig_ref] Fig 2: The difference is that Fig 5 shows [/fig_ref]. ## Random factors preserve correlations Since volatility of each time-series is normalized to 1 separately, accuracy of volatility reproduction is relative to volatilities of the underlying time-series in [fig_ref] Fig 2: The difference is that Fig 5 shows [/fig_ref] In the RFM, error in volatility is 3.1 percent of volatility with ten factors. In PCA, error is 41.7 percent of volatility with ten factors. Modeling error is here defined as the difference σ model − σ data between volatility σ model estimated from the modeled data and volatility σ data computed from the original data. Accuracy increases until 1,000 factors is reached, after which essentially no error is observed in PCA. While the RFM reproduces the overall volatility of the equity time-series faithfully, it does not capture time-dependence of volatility particularly well (data not shown). The 25th and 75th percentiles of error converge toward zero when the number of factors increases. Together these three curves form a funnel [fig_ref] Fig 3: shows statistics on the accuracy of reproduction of correlation coefficients in all... [/fig_ref] that rapidly converges toward zero. This shows that the typical accuracy of the correlation coefficient reproduction improves rapidly with the number of factors. Still, some noise persists even with the full set of 1,591 factors, for k = d. ## Correlation coefficient In PCA, median error approaches zero only with around 1,000 factors, which is largely a consequence of PCA significantly underestimating volatilities of time-series. The 25th and 75th percentiles concentrate around the median away from zero in PCA. Modeling error is here defined as the difference c model − c data between correlation c model estimated from the modeled data and correlation c data computed from the original data. Absolute modeling error is defined as the absolute difference |c model − c data |. Compared with PCA, correlation estimates in the RFM converge significantly more rapidly toward the exact value. Since error is always in the same direction in PCA, there are no differences between absolute error and relative error in PCA-based analyses. The RFM provides a more accurate description of correlation coefficients than PCA, when the number of factors is less than about 500. Noise inherent in the random factor model has the consequence that the error in correlation estimates does not disappear in the RFM even with the full set of 1,591 variables even though median estimate rapidly converges toward the observed correlation. The cross-over to regime where PCA is more accurate occurs around 500-800 factors [fig_ref] Fig 3: shows statistics on the accuracy of reproduction of correlation coefficients in all... [/fig_ref]. When the number of factors is very high, PCA gives as good as or better estimates of correlation coefficients than the RFM. A factor model with such a large number of factors is of little use in practical applications. ## Covariance The median error in covariance estimates converges rapidly toward zero in the RFM. The 25th and 75th percentiles form a funnel that converges toward zero when the number factors increases [fig_ref] Fig 4: Accuracy of covariance estimation [/fig_ref]. Despite the fact that PCA is worse than the RFM in reproducing correlation coefficients, PCA gives a better reproduction of the covariance matrix [fig_ref] Fig 4: Accuracy of covariance estimation [/fig_ref]. ## Specific volatility and explanatory power Above we saw that the RFM captured the average volatility of a time-series well. Still, PCA captured the specifics of data series more faithfully (measured by RMSE) than RFM, which suggests that specific volatility of a time-series is relatively large in the RFM. We can further elaborate on this by analyzing explanatory power. Define the explanatory power of a factor modeling by 1 − σ s /σ, where σ is the average volatility across all the securities in the data, and σ s is the average asset-specific volatility not explained by the factor model [bib_ref] The three types of factor models: A comparison of their explanatory power, Connor [/bib_ref]. While an RFM reproduces the average volatility well, the specific volatility may be higher than the average volatility. This has the consequence that an RFM may even give negative explanatory power as a consequence of the definition of explanatory power. ## Impact of the market factor The risk in the equity market is often dominated by a single factor known as the market risk factor (e.g.,. To better analyze the other possible risk factors, we subtract the first principal component, corresponding to the market risk factor, from the data and reanalyze the remaining data (the "reduced data"). [fig_ref] Fig 6: Analysis of the reduced data set in which the influence of the... [/fig_ref] shows that PCA becomes more accurate in reproducing the correlation coefficients when the impact of the market risk factor is removed from the data. Perhaps more surprisingly, reproduction of the data structure becomes equally accurate in the RFM and in PCA with respect to both error measures in the correlation coefficient [fig_ref] Fig 6: Analysis of the reduced data set in which the influence of the... [/fig_ref]. This suggests that the RFM and PCA contain equal amounts of information about the correlations. As a further check, we generated random data by sampling the normal probability distribution N(0, 1) repeatedly. that the accuracy of both the RFM and PCA is almost identical in this case. Comparison with shows that the accuracy of reproduction of the "reduced" Russell correlations does not significantly differ from the accuracy of the random data. This indicates that the fluctuations around the market risk factor are largely a product of independent "noise" contributions. Removal of the market risk factor from the data also influences the accuracy of covariance reproduction. PCA is again more accurate than the RFM in covariance reproduction [fig_ref] Fig 6: Analysis of the reduced data set in which the influence of the... [/fig_ref]. In this case, the median error of covariance matrix reproduction does not deviate from zero in the RFM, and the 25th and 75th percentiles are almost symmetrically around x-axis. ## Universality A number of probability distributions have been found useful in the random projection method [bib_ref] Dimensionality reduction by random mapping: Fast similarity computation for clustering, Kaski [/bib_ref] [bib_ref] Database-friendly random projections: Johnson-Lindenstrauss with binary coins, Achlioptas [/bib_ref]. Almost any probability distribution with zero mean, unit variance, and subgaussian tail fulfills the requirements of the Johnson-Lindenstrauss theorem [bib_ref] On variants of the Johnson-Lindenstrauss lemma, Matoušek [/bib_ref]. These findings suggest that it may not matter much which probability distribution is used in the random projections. To find out whether this is the case in an RFM, we reanalyze the data using RFMs based on six different probability distributions. We have also discussed some lowest order effects of varying the probability distribution, as well as reasons why deviation from a Gaussian distribution leads only to small corrections, in Remark 1.4 after the proof in S1 Appendix. ## Probability distributions The six probability distributions that we employ here are two sparse matrix models of [bib_ref] Database-friendly random projections: Johnson-Lindenstrauss with binary coins, Achlioptas [/bib_ref] , a column-normalized Gaussian model, a row-normalized Gaussian model, the baseline Gaussian model (defined in Sec. 2.4) and a uniform model. In each case, the probability distribution is symmetric with respect to the origin and such that the expectation is zero. Each probability distribution has a subgaussian tail. These RFMs differ from the baseline Gaussian RFM only by the construction of the random projection matrix B, and by the normalization. 4.1.1 Coin-flipping distributions. The simplest specification for random projection is the "random coin-flipping" algorithm of [bib_ref] Database-friendly random projections: Johnson-Lindenstrauss with binary coins, Achlioptas [/bib_ref]. It is defined by choosing each element B pq of matrix B randomly and independently according to rule: set B pq = +1 with probability 0.5 and set B pq = −1 with probability 0.5. The second random projection that [bib_ref] Database-friendly random projections: Johnson-Lindenstrauss with binary coins, Achlioptas [/bib_ref] proposes is based on a more sparse projection matrix defined by: set B pq = + 1 with probability 1/6, set B pq = 0 with probability 2/3 and set B pq = −1 with probability 1/6. Again each element is chosen randomly and independently of the other elements. Based on these random projections, we can define two RFMs. ## Gaussian and uniform distributions. In addition to the baseline Gaussian RFM, we analyze two different RFMs based on the normal distribution. In the first RFM, matrix B is based on the spherical uniform distribution. The elements of matrix B are defined by [formula] B ml ¼ z ml =Z;ð17Þ [/formula] where z ml � N(0, 1) are independent and Z ¼ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi P p jz pl j 2 q . In this RFM, columns of matrix B are normalized in such a way that their length is exactly one. Due to normalization of the columns of matrix B, diagonal elements of matrix B T B 2 R d�d behave as in an orthogonal matrix. Then (B T B) mm = 1 for all m = 1, 2,. . ., k. Non-diagonal elements of B T B have zero expectation and variance proportional to 1/d [bib_ref] Dimensionality reduction by random mapping: Fast similarity computation for clustering, Kaski [/bib_ref]. Hence, non-diagonal elements of B T B are approximately distributed according to zero-mean normal distribution at a relatively low dimension. Therefore B T B = 1 + �, where � 2 R d�d has nonzero elements only on off-diagonal, E½�� ¼ 0 and jE½��j < 2=d. Matrix B is then almost orthonormal. The second RFM based on the Gaussian probability distribution is a variation on the theme: Instead of column-normalization in the first model, rows of projection matrix B are normalized to unit length. This is the only difference between the two RFMs, but it is sufficient to require a different normalization constant. The sixth considered RFM is defined by projection matrix B, which is based on the continuous uniform probability distribution. Each element in the projection matrix B is chosen randomly and independently from the uniform distribution on interval [−1, 1], that is, B mn � U(−1, 1) for each m, n. The results suggest that the details of how the projection matrix is specified are not that important. Almost any sufficiently regular construction of the random projection matrix (when properly normalized) produces a factor model, which preserves the approximate correlation structure. The main requirement here seems to be that matrix elements are chosen randomly and independently of other matrix elements. This supports the view that the RFM represents quite well how the bulk of factor models would reproduce the analyzed set of timeseries. ## Universality of distributions # Discussion ## Random factors as explanatory factors We set out to analyze the impact of random selection of factors on a linear factor model. Our interest was in whether and how randomness in the choice of factors impacts the reproduction of long equity time-series in a factor model, and the preservation of their interdependence. We found that a random factor model (1) provides an almost orthonormal, non-multicollinear set of factors; (2) preserves the correlation matrix well; (3) preserves average volatility well; (4) has well-defined theoretical bounds on accuracy of the model; (5) preserves the structure of the data but not necessarily the details of single time-series. These are novel findings. One can confidently say that the random factor models performs significantly better than one would assume. At the same time, it is worth pointing out the weak points of an RFM: (1) explanatory power of an RFM is quite low; (2) RMSE error of reproduction is higher than in PCA; (3) Only in the limit of infinite number of factors, an RFM is ensured to reproduce the original timeseries perfectly; (4) an RFM does not provide factors capable of giving intuitive economic explanation of the data. It may seem unlikely that a factor model with randomly-chosen factors could be used for any kind of factor modeling. One of the reasons for the ability of an RFM to capture the details of an equity time-series resides in the fact that random factors are, as a consequence of independence of elements, almost orthogonal to each other. The number of almost orthogonal vectors is higher in a higher-dimensional space, often attributed to., which reduces the impact of the "curse of dimensionality" [bib_ref] Approximate nearest neighbors: towards removing the curse of dimensionality, Indyk [/bib_ref] and thereby makes data representation more feasible. A suitably high number of random factors will then span a subspace sufficient to capture the return time-series at the desired accuracy. If one were to try to model a set of time-series with a factor model with non-informative factors, the results would likely resemble those obtained with a random factor model. This obviously assumes that the set of factors would have low cross-correlations and little autocorrelation. Then our results would give an idea of the accuracy that one would reasonably expect to observe in the resulting model. In other words, our results give a lower bound on the accuracy of an informative factor model. ## Universality In a classical factor model, only a few factors are statistically significant. Then, explanatory power of each factor should be large. In a statistical factor model, a larger number of factors is often used, which has the consequence that a larger ambiguity in the choice of factors is encountered [bib_ref] Honey, I shrunk the sample covariance matrix, Ledoit [/bib_ref] , as several different sets of factors may provide almost equally good fit to the data. In an RFM, each factor has only a small explanatory power, which suggests that a large number of factor sets provide essentially equally good descriptions of the data and its structure. This was observed in our computational experiments. The number of random factors seems to be more important than fine-tuning of random factor time-series. The way an RFM is constructed is not important as long as the elements of projection B are independently drawn from a suitably regular probability distribution with zero expectation and subgaussian tails. We obtained almost identically accurate results regardless of the probability distribution used. These findings suggest that a kind of universality of RFMs is present, at least with respect to correlation coefficients. The results are largely dominated by a set of typical RFMs that have a rather similar accuracy of data reproduction. We have called this set of factor models the bulk. The analysis of the proof of Theorem 1.1 in S1 Appendix (see Remark 1.4 in S1 Appendix) supports the view that universality is present with respect to probability distributions. The assumption that probability distribution is Gaussian is not necessarily required in the theorem. It suffices to assume independence of the random matrix elements, and it is likely that this requirement can be relaxed further. # Conclusions A random factor model captures average volatility and reproduces correlations of the data with well-defined probabilistic error bounds, despite the fact that the factors are random and they are in no way fitted to the data. In the limit of large number of factors, the random factor model converges toward the exact reproduction of the data. Random factor modeling answers the question: how many factors does a generic linear factor model require to describe the correlation matrix of a dataset at a specific accuracy while preserving the average volatility of the time-series. Random factor models show universality of results: regardless of the method used to generate random factors, the results were essentially equally good. Supporting information S1 Appendix. Accuracy of the random factor approach. In S1 Appendix, we prove the main results used in the text for the mean and variance of the projection operators involved in the random factor models. We are mainly interested in controlling their asymptotic dependence for large values of the dimensions k and d of the factor matrix. (TEX) [fig] Fig 1: provides three examples of equity time-series reproduction using the RFM and PCA. The RFM (grey solid curve in)provides a good reproduction of the single time-series even with a low number of factors. The accuracy of the reproduction improves with the number of factors: the agreement of the RFM and the data is very good with 500 factors. [/fig] [fig] Fig 3: shows statistics on the accuracy of reproduction of correlation coefficients in all analyzed pairs of stocks. In the RFM, the median error converges rapidly to zero with only a few factors. [/fig] [fig] Fig 4: Accuracy of covariance estimation. Median error (left panel; solid gray curves; measure in percentage points) in covariance in all pairs in the data-set estimated using 1,000 different random factor models, together with 25th and 75th (dashed gray curves) percentiles of error in covariance estimates. The results are compared with the estimates of covariance based on PCA (solid black curve), together with the 25th and 75th (dashed black curves) percentiles. The results are shown as a function of the number of factors (abscissa). Median absolute error (right panel; solid gray curves; measure in percentage points) in covariance in all pairs in the data-set estimated using 1,000 different random factor models, together with the 25th and 75th (dashed gray curves) percentiles of error. The results are compared with the estimates of covariance based on PCA (solid black curve), together with the 25th and 75th (dashed black curves) percentiles. https://doi.org/10.1371/journal.pone.0206551.g004 [/fig] [fig] Fig 2: The difference is that Fig 5 shows (one minus) the ratio of the residual volatility and the total volatility, while Fig 2 shows that the RFM captures the time-averaged volatility well. Explanatory power of the RFM clearly increases with the number of factors, even when the number of factors increases toward the number of time-series. [/fig] [fig] Fig 6: Analysis of the reduced data set in which the influence of the market is removed. Error and absolute error in correlation coefficient and in covariance estimates in an random factor model (gray curves) and in PCA (black curves). Solid lines are median estimates, dashed lines 25th and 75th percentiles. https://doi.org/10.1371/journal.pone.0206551.g006 Fig 7. Reproduction of correlation coefficient in randomly-generated data. Error in correlation coefficient reproduction (left panel) of the random data using the random factor model (dashed gray curve) and PCA (black solid curve). Curves are shown as functions of the number of factors. Differences are in percentage points. The right panel shows absolute error in correlation reproduction of the random data using the random factor model (dashed gray curve) and PCA (black solid curve). https://doi.org/10.1371/journal.pone.0206551.g007 Random factors preserve correlations [/fig] [fig] Fig 8: shows that all six RFMs produce almost equally accurate results. To reduce noise, shows results averaged over 50 sample runs. When the number of factors exceeds 10, all RFMs produce almost identical median accuracy. The only deviation is the column-normalized Gaussian model, which deviates from the other RFMs when the number of factors is less than 5. All the other RFMs produce identical results also in this regime. The accuracy of the 25th and 75th percentiles mainly depends on the number of factors, not much on the way factors are generated or on the underlying probability distribution. [/fig] [table] Table 1: The central parameters used. Number of data points in each time-series N Number of time-series, e.g., equities k Number of factors https://doi.org/10.1371/journal.pone.0206551.t001 Random factors preserve correlations PLOS ONE | https://doi.org/10.1371/journal.pone.0206551 December 21, 2018 [/table]

Motor intervention with and without Nintendo® Wii for children with developmental coordination disorder: protocol for a randomized clinical trial Background: Despite the benefits highlighted by motor interventions based on virtual reality for children with Developmental Coordination Disorder (DCD), there are still doubts as to whether these are greater than those obtained with conventional interventions due to the absence of systematized protocols, and lack of evidence. Here, we present a protocol to systematically compare the effects of two motor-training programs (one Nintendo® Wiibased and the other no-Wii motor activities) on the motor learning in children with DCD. Methods/design: Two intervention protocols (one based on Nintendo® Wii and the other no-Wii motor activities) will be carried out, with interventions occurring twice a week in 60-min sessions, with a minimum of 12 and a maximum of 16 sessions per child. The protocols were developed based on the domains of the Movement Assessment Battery for Children -Second Edition (MABC-2) (Manual Dexterity, Aiming and Catching, Balance), with two activities for each of the MABC − two domains. The study will include children aged 7 to 10 years with a total MABC-2 score ≤ 16, and a Developmental Coordination Disorder Questionnaire (DCDQ) score < 46 (age of 7 years), score < 55 (age group of 8 to 9 years and 11 months), or score < 57 (age of 10 years) as scored by the parents. Children will be randomly allocated by draw in one of the two intervention protocols. MABC-2 and DCDQ will be applied before and after intervention to evaluate the effects of the interventions on motor performance and parents' perception, respectively. Motor learning will be assessed by means of the scores obtained in the games. Evaluators and therapists will be trained and evaluators will be blind regarding the data of the children in the study.Discussion: Owing to its motivating aspects, training with Nintendo® Wii may be particularly beneficial for children with DCD. The results of this study protocol should help researchers and therapists to better understand the benefits of Nintendo® Wii-based motor intervention over those obtained with no-Wii interventions in children with DCD. It should also create references about more systematized protocols for replication in clinical practice, seeking the improvement of the motor components of these children. Trial registration: RBR-89ydgj # Background Children with compromised motor control are widely mentioned in various studies [bib_ref] Action planning and position sense in children with Developmental Coordination Disorder, Adams [/bib_ref] [bib_ref] Directionspecific impairment of stability limits and falls in children with developmental coordination..., Fong [/bib_ref] [bib_ref] Children with developmental coordination disorder demonstrate a spatial mismatch when estimating coincident-timing..., Caçola [/bib_ref] , due to the fact that affected motor ability directly or indirectly affects the performance of functional activities. Developmental Coordination Disorder (DCD) has a number of characteristics related to motor development that have a meaningful impact on the daily and school life of many children [bib_ref] Developmental coordination disorder and its cause: The road less travelled, Wade [/bib_ref]. DCD diagnostic criteria involve significant motor alterations, compromising daily life, school life or leisure activities, and the fact that these alterations appeared in the earlier stages of children's life despite sufficient practice of motor activities. Neuroimaging data have highlighted important cortical alterations in children with DCD, specifically in the frontal, parietal, and temporal regions [bib_ref] Dysfunction of the attentional brain network in children with Developmental Coordination Disorder:..., Querne [/bib_ref] [bib_ref] Parietal dysfunction in developmental coordination disorder: a functional MRI study, Kashiwagi [/bib_ref] during the performance of manual tasks, which may be associated with a slower processing of motor information in these children when compared to typical ones [bib_ref] Neural Correlates of Developmental Coordination Disorder: A Review of Hypotheses, Zwicker [/bib_ref]. Such restrictions may lead to reduced social participation [bib_ref] Cortical functioning in children with developmental coordination disorder: a motor overflow study, Licari [/bib_ref] and school performance [bib_ref] Social participation by children with developmental coordination disorder compared to their peers, Sylvestre [/bib_ref] meaningfully, since many children with DCD tend to isolate themselves from other children because they cannot perform motor activities at the same pace as their peers due to the limitation in information processing. They also tend to become more anxious and insecure, and feel more motivated to perform activities when they are alone, as well as those with sedentary characteristics [bib_ref] Physical and mental health of children with developmental coordination disorder, Caçola [/bib_ref]. Motivating strategies in the intervention with these children seem to be the key to success in terms of participation and functional gains [bib_ref] Longitudinal examination of objectively-measured physical activity and sedentary time among children with..., Kwan [/bib_ref]. The use of technology has gained prominence in recent years among the strategies adopted since resources, such as interactive games based on virtual reality (VR), offer instant feedback and a larger number of repetitions of body movements per session than many conventional motor intervention techniques, such as physiotherapy, occupational therapy, cognitive orientation to daily occupational performance (CO-OP) or Neuromotor Task Training (NTT) [bib_ref] Understanding physical activity and motivations for children with Developmental Coordination Disorder: an..., Kwan [/bib_ref]. This facet may promote greater efficacy of the motor intervention in these children. More specifically, the use of the Nintendo® Wii in the rehabilitation of children with motor alterations has been promoted for ease of measuring the force applied and capturing pressure change available in the Wii Balance Board (WBB), besides the fact that the Wii motion control allows great stimulation of the hand-dexterity motor component [bib_ref] Virtual reality use in motor rehabilitation of neurological disorders: a systematic review...., Rahman [/bib_ref] [bib_ref] Video capture virtual reality as a flexible and effective rehabilitation tool, Weiss [/bib_ref]. Balance, Aiming and Catching are the domains of the Movement Assessment Battery for Children , that are regarded as the gold standard for the identification of DCD in children [bib_ref] Virtual reality as a therapeutic modality for children with cerebral palsy, Snider [/bib_ref]. These aspects make the Nintendo® Wii very useful as a VR resource for motor interventions in children with DCD. At present, however, little evidence exists in the literature on comparisons between the benefits of VR training, such as Nintendo® Wii, and conventional interventions such as without Wii [bib_ref] Can the Movement Assessment Battery for Children-Test be the "gold standard" for..., Venetsanou [/bib_ref] [bib_ref] The efficacy of two task-orientated interventions for children with Developmental Coordination Disorder:..., Ferguson [/bib_ref] [bib_ref] The impact of Wii Fit intervention on dynamic balance control in children..., Jelsma [/bib_ref]. The handful of existing studies that compare interventions with and without VR for children with DCD still show limitations about evidence created by the VR intervention [bib_ref] The efficacy of two task-orientated interventions for children with Developmental Coordination Disorder:..., Ferguson [/bib_ref] [bib_ref] Effect of Wii-intervention on balance of children with poor motor performance, Mombarg [/bib_ref] [bib_ref] Low-cost virtual reality intervention program for children with developmental coordination disorder: a..., Ashkenazi [/bib_ref] [bib_ref] An investigation of the impact of regular use of the Wii Fit..., Hammond [/bib_ref] [bib_ref] A crossover randomised and controlled trial of the impact of active video..., Straker [/bib_ref]. Moreover, it is not clear whether one type of intervention offers greater gains than the other due to the shortcomings from the scarce previous studies already published. Based on a recent systematic review by Hickman et al. [bib_ref] Use of active video gaming in children with neuromotor dysfunction: a systematic..., Hickman [/bib_ref] evidence for advantages gained by a VR intervention for children with DCD are not consistent in relation to conventional therapy. The authors argued that due the heterogeneity of assessment tools and outcomes fair comparisons were not possible. So, we believe that in this study we might see fair comparisons because the tasks will be closely matched by motor domain target into the training protocol. These limitations of present approaches hinder a wellinformed conclusion as to whether training with VR is more effective than conventional training in children with DCD. In this paper we present a protocol for a randomized clinical trial to systematically compare two motor-training programs (with and without Nintendo® Wii) on motor learning in children with DCD. # Methods/design ## Design and general characteristics The protocol consists of a clinical, randomized controlled, blinded trial with two arms: training with the Wii, and conventional training without the Wii. Therapists will undergo instructional training to become familiar with all protocol activities, as well as with the scoring system of each game. The instructional training will occur a month before the beginning of the trial, which has a total duration of 8 h, for 2 h weekly. Despite the rigidly structured task settings required over the sessions, the therapists will be allowed to display their skills as therapist in order to promote a motivational environment for the children during the sessions as well as a great relationship between therapist and patient. There will be different therapists in each of the two intervention protocols, professionals from the areas of physiotherapy, physical education, and occupational therapy. The therapists in the two groups have comparable backgrounds and skill sets. All therapist teams have the same experience with pediatric rehabilitation, around 4 years of experience in this field. Children fulfilling the inclusion criteria in the study will be randomly allocated by draw into one of the two intervention groups. The flow chart of the subjects which will be allocated in the study is shown in [fig_ref] Figure 1: Flow contra-indications such as high levels of anxiety, impulsivity or attentional difficulties [/fig_ref] (Additional file 1). ## Patient population Participants will be children aged from 7 to 10 years who have been diagnosed with Development Coordination Disorder. They will be recruited from public and private elementary schools in the city of São Carlos, State of São Paulo, Brazil. The recruitment process as well as advertising of our study will be based on meetings at schools. If necessary, we will use social media to advertise the project and increase the number of potential children with DCD. Their diagnosis is based on the following Diagnostic and Statistical Manual of Mental Disorder -V (DSM-V)criteria for DCD: Criteria A -Children displaying motor performance regarded as low for the age range and for the conditions of practice opportunity. The Movement Assessment Battery for Children -Second Edition (MABC-2) will be used for the evaluation of their motor performance. They will be allocated in the Amber Zone range for DCD with a total score percentage of ≤ 16, or in the Red Zone range with a total score percentage of ≤ 5, to be included in the study. Criteria B -The limited motor performance of the children interferes significantly with daily life, school life, and leisure activities. The Developmental Coordination Disorder Questionnaire (DCDQ -Brazilian version) [bib_ref] Cross-cultural adaptation of the Developmental Coordination Disorder Questionnaire for brazilian children, Prado [/bib_ref] will be used to assess, according to the perspective of the parents/caregivers, the loss in children's functional life, in addition to direct interviews with them about the children's routine. Criteria C -Symptoms appeared in the initial developmental stages of the children. Direct interviews with parents/caregivers will be carried out to ascertain such characteristics. Criteria D -The motor limitations showed by the children do not come from intellectual or visual impairment, or neurologic conditions such as cerebral palsy, dystrophy or any degenerative disease. In order to eliminate these associated conditions, school data about the children will be assessed, teachers will be interviewed and also physiotherapists will offer assessments in order to eliminate any sign of the mentioned neurologic conditions. In addition, psychiatric disorder history will be assessed through the parents' interview as well as based on children's forms at school in order to avoid the heterogeneous profile of those children. If we find children who have already received a psychiatric diagnosis (other than DCD) we will exclude them from the study. In addition, we will exclude children who may have Highly anxious, inattentive or oppositional children who might be unable to complete either intervention program will be excluded from the study because they might find the interventions distressing. Children will be allowed to continue regular physical education classes at schools. Other concomitant care or interventions during the period of intervention will not be allowed. In case any children are missed before the intervention ends, we use the intention-to-treat analysis. ## Interventions Both intervention protocols are based on the Movement Assessment Battery for Children -Second edition (MABC-2) domains: Manual Dexterity, Aiming and Catching, and Balance. Six games/activities for Nintendo Wii are used that target these domains and six no-Wii activities that are compatible with the activities selected for Wii. The reason for selecting these six of activities is that they address potential improvements in the skills that are required for the standard assessment for children with DCD. The second reason was to render the games/activities as close as possible to those required for MABC-2 evaluation in terms of movement standard. Each game/activity will continue for 7 min and in total 42 min will be spent on the six activities. With the purpose of exchanging materials and equipment from one game to the next, thus allowing to complete the total time of 60 min per session. Sessions of both protocols will be performed twice a week, with duration of 60 min per session, and with a minimum of 12, and a maximum of 16 training sessions in total. The first session will familiarize the children with the games/activities. The second sessions consists of a pre-test and the last the post-test for motor learning outcome. The number of mistakes, hits, and respective score will be counted in each one of the games/activities. Pre-post scores for each training condition will be compared. The two protocols each have six activities. For the protocol with Wii, existing games will be use and for the no-Wii protocol 'real life' activities will be used that target the same domains as the Wii tasks. The total score for each session will be the sum of the points obtained in all the attempts performed. The sequence of the games will be randomized, with a selection among 16 possible combinations, considering two activities for the same domain in sequence in all of these combinations [fig_ref] Table 2: Training activities with Wii and the three domains [/fig_ref]. ## Experimental protocol with wii This protocol was based on percepto-motor activities, consisting of activities and games based on VR, supported by Nintendo® Wii resources: the Wiimote control and the WBB platform, communicating via bluetooth. The training activities are detailed below: Frisbee This activity will be performed with the children holding the Wiimote control in the fist, in a space of 1.10 m 2 , at a 2-m distance from the television screen to which the Nintendo® Wii will be connected, performing throws of the virtual Frisbee, aiming to hit the target in the scoring area projected. Children will be encouraged to perform the highest number possible of throws in the period of 7 min, trying to hit the scoring areas worth 10, 50, and 100 points. Every time the Frisbee hits these spaces, it will count as a hit and as a mistake each time it hits outside them. The sum of all attempts hitting any scoring area during 7 min in each session will be the basis of the score. ## Table tennis This activity will be performed with the children holding the Wiimote remote control in their fist, and they will be encouraged to use it as if it were a table tennis paddle, to serve, hit and hit the ball again, trying to score more points than the opponent (virtual opponent chosen by the machine). Each game will automatically end when one of the players scores 8 points. Each point scored by the children will count as a hit and those scored by the opponent (machine) as a mistake. The children must score the maximum number of points in 7 min. ## Bowling This activity will be performed with the Wiimote control held in the fist, which serves as if it were the bowling ball. The aim is to perform the throws and try to knock down the highest number of pins. Two blocks of nine attempts will be performed. At the end of the 10th block, children will start a new session of two attempts each until the end of the 7-min period. Each throw knocking pins down will count as a hit, or as a mistake in the opposite case. Children who knock down all 10 pins at once will be considered Strikers and will double the score for the next throw, while those knocking down the 10 pins in two throws (first to ninth blocks) or three (10th block) will be considered a Semi-Striker, receiving half of the score they achieve in the next throw as a surplus. Archery This activity will be performed with the Wiimote remote control held in the children's fist. The movement to be performed with the Wiimote should represent the action of a bow moving the arrow to reach the aim with different scoring areas, varying from 1 to 10 points. The children, in the figure of an avatar, will go through three stages of the game at the beginner level, each one with different scenery and distances (10 m, 25 m, and 35 m) between avatar and aim. At the end of the three stages of attempts, children will return to the beginning stage and continue to perform new attempts until the end of the 7-min period. Each arrow shot and sticking to the target will count as a hit, and as a mistake in the opposite case. The total score for each session will be the sum of the points obtained in all the attempts performed. ## Tightrope walk This activity will be performed with the WBB equipment, fixed to the ground, in a space of 1.10 m 2 and at a distance of 2 m from the television screen. Children will have to step with both feet on the equipment and perform the action of the game, consisting in a 35-m tightrope walk between the top of virtual buildings. As a form of equalizing scores, the total length of the rope is divided in three parts: the two first of 12 m each and the last of 11. The basic movement that children will perform is raising their feet alternately, moving the trunk sideward so that the avatar maintains the balance as it walks on the rope. Each third part of the rope walked without falling will count as a hit, while any fall in lengths shorter than 12 or 11 m will count as a mistake. The first 12 m walked will be worth 15 points, the 12 intermediary meters will be worth 20, and the last 11 m 10 points. ## Marble balance This activity will be performed with the WBB equipment fixed to the ground, similar to what described for the tightrope walk. Children will move their trunk to different positions while trying to throw the ball in the holes of the virtual platform, which will be turning all the time, provoking greater unbalance and difficulty in the game. Every ball falling out of the virtual platform will count as an error, and each one in the respective hole will be worth a point. As the balls disappear, the children will move to a new platform, with more balls to be thrown into the holes. Scores will be calculated by the Nintendo® Wii equipment itself with basis on the performance of the avatar commanded by the children. ## Experimental protocol no-wii training This protocol was based on a proposal of perceptomotor type, and named no-Wii training. Its training protocol is detailed on the following table: ## Frisbee The game of Frisbee consists in throwing a Styrofoam disk measuring 24 cm of circumference [fig_ref] Figure 2 a: Frisbee, b Frisbee target [/fig_ref] on a paper card target with a circumference of 2.82 m and placed at a distance of 4 m [fig_ref] Figure 2 a: Frisbee, b Frisbee target [/fig_ref]. The target must be fixed to the ground and display three different scoring areas (10, 50, and 100 points). ## Table tennis The game of table tennis will be performed with the elevation of one of the two halves of the table, allowing children to throw and hit the ball again with their paddle [fig_ref] Figure 3: Tennis table [/fig_ref]. The aim of this activity consists in making the ball hit the raised half of the table after it hits the horizontal part and going back to the same side, and children will be given a hit for each movement of this kind. Balls falling out of the game area, multiple rebouncing on the horizontal part of the table and a serving or reception error will count as mistakes. ## Bowling The game of bowling will be performed with the use of 10 PET bottles containing 600 ml of water each [fig_ref] Figure 4: PET bottles and ball in the bowling target area [/fig_ref] , placed at a distance of 4.83 m for children aged 7 and 8 years, and at a distance of 6.03 m for those aged 9 and 10 years. The aim of the activity is to throw a 0.5-kg medicine ball towards the bottles and knock down the highest number possible at once [fig_ref] Figure 4: PET bottles and ball in the bowling target area [/fig_ref]. Similar to conventional bowling, children will be conceded two throws per block of attempts, considering that in the 10th block the children will have the right to three attempts. The bottles knocked down in the first attempt of each block will be put in place again only after the children have concluded the throws in the second or third attempt (only for the 10th block). Each bottle knocked down will be worth a point. Throws failing to knock down pins will count as zero in the score and as mistakes, while successful attempts will be regarded as hits. If children manage to knock down all bottles at once, they will achieve the Striker score, doubling it in the following attempt, and if they knock down all the bottles in two attempts of a block or in the three attempts of the 10th block they will attain the Semi-Striker score, receiving half the score of the following throw as surplus. ## Bow and arrow This activity will be performed with a plastic bow-andarrow commercial set [fig_ref] Figure 5 a: Bow and arrow, b Target for bow and arrow [/fig_ref] , including a 62-cm bow and the arrows, each one of 41 cm. The arrows end with a suction cup which sticks to the target [fig_ref] Figure 5 a: Bow and arrow, b Target for bow and arrow [/fig_ref] , to indicate the score that each child achieves in the game. A wooden target, covered with an adherent plastic surface so that the suction cups at the tip of the arrows stick to it, will be used. It measures 41 cm in circumference, and it will be fixed to the wall with a 120-cm adjustable rope in front of the children, at their visual height, at a distance of 2.30 m for 7-and 8-year-old children and 2.97 for children aged 9 and 10 years. Each arrow hitting the target and sticking to a scoring area will count as a hit, while those not fixing to it as a mistake. Scores will be based on the scale displayed on the target itself, ranging from 1 to 10 points. ## Balance disk This activity will be performed with a vinyl balance disk measuring 40 cm in circumference [fig_ref] Figure 6: Balance diskTable 4Sequence of movement for balance disk Sequence of movements -Balance... [/fig_ref] placed on the ground; the children will perform 14 different static balance postures on top of it . The postures were based on, and adapted from, the Berg Balance Scale [bib_ref] Measuring balance in the elderly: development and validation of an instrument, Berg [/bib_ref]. Children aged 7 and 8 years must maintain the postures for 15 s, while 9-and 10-year-old children must keep them for 30 s. Each posture performed correctly and for the time due will count as a hit, while unbalances, falls, stepping on feet or putting hands on the ground will be regarded as mistakes. In the second case, children must go to the following posture. Each hit will be worth 10 points, and at the end of the 14 postures, they will repeat the same sequence until the time is over. ## Balance beams This activity will be performed with three 3 m-long wooden beams, measuring 3 cm in height, parallel to the ground [fig_ref] Figure 7: Balance beams [/fig_ref] , each one with a different width, therefore offering children various degrees of difficult. The widest stick measures 6 cm, the intermediary 4.5 cm and the narrowest 3 cm. Three wooden traverses measuring 15 × 1.5 × 5 cm will be placed at the beginning of each beam, 50 cm apart from one another. Children will have to move on each one of the beams, from the widest to the narrowest, stepping with one foot at a time, so that the heel of the front foot is close to the toes of the back foot. Each beam stepped correctly will count as a hit, and any fall, using the beam or the ground as a support, slipping or losing contact of the feet with the beam, touching the ground or not performing the steps with feet close will count as an error. Scores for beams stepped correctly are as follows: 10 points (6-cm wide beam), 15 points (4.5-cm wide beam) and 20 points (3-cm wide beam). At every unsuccessful attempt, children will go back to the beginning of the beam where the mistake occurred and start the route again, moving to the next beam after doing the correct movement on the previous one. ## Outcomes measures All tests will be applied by trained evaluators, blind regarding the allocation of the children in the intervention groups. Evaluators will be professionals in the areas of physiotherapy, physical education, and occupational therapy. Each child will be assessed by the same person before and after treatment. ## Primary outcome measure The primary outcomes of this study is the motor performance Motor performance -MABC-2 The motor performance of the children will be assessed through the MABC-2 total standard score (TSS). As mentioned before, MABC-2 is considered a gold standard tool to identify motor performance problems in children from 3 years old. MABC-2 is divided in three different age bands: the band 1 for children aged from 3 to 6 years old; band 2 for children aged from 7 to 10 years old; and band 3 for children aged from 11 to 16 years old. There are eight tasks in total, three for the Manual Dexterity domain, two for the Aiming and Catching domain; and three for the Balance domain. Children received a specific score in each domain and their motor performance classification is based on total score and percentiles values. Children < 5°percentile present motor difficult; ## Secondary outcomes measures motor learning Motor learning in children will be assessed by means of the score obtained in the games. Individual scores in each of the six games from the second session (pre-test) will be summated as well as those from the six games of the last session (post-test) to obtain the general measurement of motor learning due to the treatment. Individual scores in each game and for domain will also be counted (Manual Dexterity, Aiming and Catching, and Balance), before and after intervention. With the purpose of analyzing whether some activities are better learned in one type of intervention than in another. A specific form will be available for each therapist fills this information in each session. ## Signs indicating dcd -developmental coordination disorder questionnaire (dcdq) The DCDQ [bib_ref] Cross-cultural adaptation of the Developmental Coordination Disorder Questionnaire for brazilian children, Prado [/bib_ref] will be used with parents/caregivers of the children, who will fill in the questionnaire before they begin the treatment and also after the end, seeking to verify possible changes in parents' perception about the motor characteristics of the children. We expect that the parents will fill the DCDQ in about 10-15 min. ## Anthropometric measurements On the same day of the MABC-2 pre-test evaluation, anthropometric data about BMI (Body Mass Index), body fat percentage (BF%) and waist circumference (WC) of the children will be collected. Weight measurements for BMI will be made with a portable digital W602 (WISO®) scale, and height will be measured with a portable Wood (WCS®) stadiometer. BMI will be calculated through the height/weight relation [bib_ref] Indices of relative weight and obesity, Keys [/bib_ref]. To calculate the BF% of the children, measurements around triceps and the subscapular skinfold will be done with a Lange® adipometer. Waist circumference will be measured in the narrowest region between the last rib and the umbilicus with a Sanny® metric tape. All anthropometric measurement collection procedures will be done in the observance of the techniques standardized by the literature. The anthropometric measurements will be performed in about 10-15 min. ## Motivation -enjoyment scale At the end of the last session, the perception of satisfaction with each one of the activities and with the intervention as a whole will be assessed for each child through the Enjoyment Scale, an adapted scale by Jeslma et al. [bib_ref] The impact of Wii Fit intervention on dynamic balance control in children..., Jelsma [/bib_ref] translated to Portuguese. The score is expressed by the number of smileys, ranging from 0 (no fun at all) to 4 (super fun). Using this scale, each therapist will have direct results about the impact that the activities and the treatment as a whole had on each child. The Enjoyment Scale will be performed in about 5 min. # Ethical approval The respective research project of the present interven- ## Sample size Sample size was calculated using the G*Power Software (3.1.9.2 version, Germany) based on a pilot study (n = 6). The calculation was performed considering the withingroup comparisons for the Wii group, a power of 80%, and an alpha of 5%. The MABC total score assessed before and after 12 sessions was considered the main outcome and then considered for the analysis. The effect size was 0.80 (baseline: 58.0 ± 8.7; post 12-session intervention: 65.5 ± 9.9). Therefore, a sample of 15 participants was required for each group. In order to account for possible dropouts, we increased the sample to 16. ## Randomization Children will be randomly distributed by draw into one of the two training groups within a week at most after MABC-2 assessment. In case the draw leads to a sequence of four children in the same treatment group, the following four will be automatically allocated in the other. Once the distribution of blocks of four children is equalized, a new draw will be done for the following children to be randomly distributed. A person not involved with the study will receive a list of all children and will write in one piece of paper the term "Wii" and in another piece of paper the term "no-Wii." This person will fold each piece of paper and will request to another collaborator not involved in this study to choose between the two papers. Then, following this draw, each child will be allocated into Wii or the no-Wii intervention group. ## Blinding Assessors will be blinded about children's allocation group. In this sense, the draw for children randomization will be carried out by a professional not involved in the assessments and interventions of the project. Blind statistical analyses will be performed, having children's names and intervention groups modified by numeric codes in the spreadsheets. # Statistical methods At first, descriptive analysis will be done with the distribution of relative and absolute frequencies, averages, standard deviations, medians, mode, and minimum and maximum number in order to characterize children. All data will be inserted into spreadsheets in the SPSS statistical package, version 20.0 for Windows. Next, analyses regarding normality and homogeneity tests will be performed. There will be two independent groups because of the design of the study, and there will also be intergroup comparative analyses in each one, constituting the results of motor performance and learning evaluation pre test and post test, as well as the comparisons between such intergroup evaluations. In case of parametric distribution, analyses will be performed through analysis of variance (ANOVA) and analysis of co-variance (ANCOVA) utilizing the Bonferroni correction. In case of non-parametric distribution, intergroup comparative analyses will be carried out by means of the Mann-Whitney U test and intergroup comparison Description of screening, assessment, and intervention phases related to the study through the Wilcoxon test. The significance level adopted will be p < 0.05 for all measurements. In order to verify the dimension of the effect of the interventions realized, the Cohen test will be applied with the following reference values: d = 0.3 indicates a small dimension of the effect, while d = 0.5 and d = 0.8 indicate a moderate and a large one [bib_ref] Using effect size-or why the P value is not enough, Sullivan [/bib_ref] , respectively. ## Study organization The present study will be performed at the Physiotherapy Department of the Universidade Federal de São Carlos (DFisio/UFSCar), in the city of São Carlos, São Paulo, Brazil. Municipal and State Education secretaries of São Carlos are partners in this project, since they authorized the access of researchers to the schools to recruit the children target of this study, which counts on the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), with process number 2015/24291-0. There is no other role of this funder in the design of this study. A data and safety monitoring committee is not required in this study. The main information regarding screening, assessments, and interventions is available in . # Discussion This study protocol is the first to directly compare the efficacy of two motor-training protocols in children with DCD. The protocol is based on the domains of the MABC-2 domains. Various studies have already demonstrated the efficacy of intervention programs based on VR for these children [bib_ref] The efficacy of two task-orientated interventions for children with Developmental Coordination Disorder:..., Ferguson [/bib_ref] [bib_ref] The impact of Wii Fit intervention on dynamic balance control in children..., Jelsma [/bib_ref] [bib_ref] Effect of Wii-intervention on balance of children with poor motor performance, Mombarg [/bib_ref] [bib_ref] Low-cost virtual reality intervention program for children with developmental coordination disorder: a..., Ashkenazi [/bib_ref] [bib_ref] An investigation of the impact of regular use of the Wii Fit..., Hammond [/bib_ref] [bib_ref] A crossover randomised and controlled trial of the impact of active video..., Straker [/bib_ref] [bib_ref] Rationale, design and methods for a randomised and controlled trial of the..., Straker [/bib_ref] [bib_ref] Effect of training children with Developmental Coordination Disorders in a virtual environment..., Ashkenazi [/bib_ref] [bib_ref] Randomised controlled trial of referral to a telephone-based weight management and healthy..., O&apos;brien [/bib_ref] [bib_ref] Children with developmental coordination disorder play active virtual reality games differently than..., Gonsalves [/bib_ref] [bib_ref] Motor learning: an analysis of 100 trials of a ski slalom game..., Smits-Engelsman [/bib_ref] [bib_ref] A sensorimotor approach to the training of manual actions in children with..., Snapp-Childs [/bib_ref] , but the absence of further protocol systematization has been an important hindrance for professionals and researchers in this field to clearly understand the impact of such interventions on children's activity performance. Studies comparing interventions based on VR and conventional interventions [bib_ref] The efficacy of two task-orientated interventions for children with Developmental Coordination Disorder:..., Ferguson [/bib_ref] [bib_ref] Low-cost virtual reality intervention program for children with developmental coordination disorder: a..., Ashkenazi [/bib_ref] [bib_ref] An investigation of the impact of regular use of the Wii Fit..., Hammond [/bib_ref] [bib_ref] A crossover randomised and controlled trial of the impact of active video..., Straker [/bib_ref] are scarce, and their results tend to demonstrate that despite the benefits of VR, conventional interventions seem to be more effective in the improvement of motor performance of children with DCD [bib_ref] The efficacy of two task-orientated interventions for children with Developmental Coordination Disorder:..., Ferguson [/bib_ref] [bib_ref] Low-cost virtual reality intervention program for children with developmental coordination disorder: a..., Ashkenazi [/bib_ref] [bib_ref] An investigation of the impact of regular use of the Wii Fit..., Hammond [/bib_ref] [bib_ref] A crossover randomised and controlled trial of the impact of active video..., Straker [/bib_ref]. Due to limitations and differences between the protocols adopted, though, these results must be interpreted carefully. Hence, when testing the efficacy of these two type of intervention programs, planned in similar manner, with similar scoring systems and activities, it will be possible to enhance evidence about the benefits in motor performance and learning of one program over the other. One documented advantage of VR training, such as the Wii intervention, is the motivating aspects because the projected architecture on the environment of VR is capable of hold the children's attention, being an important aspect for rehabilitation of children with DCD [bib_ref] The impact of Wii Fit intervention on dynamic balance control in children..., Jelsma [/bib_ref]. [fig] Figure 1: Flow contra-indications such as high levels of anxiety, impulsivity or attentional difficulties. [/fig] [fig] Figure 2 a: Frisbee, b Frisbee target [/fig] [fig] Figure 3: Tennis table [/fig] [fig] Figure 4: PET bottles and ball in the bowling target area [/fig] [fig] Figure 5 a: Bow and arrow, b Target for bow and arrow [/fig] [fig] Figure 6: Balance diskTable 4Sequence of movement for balance disk Sequence of movements -Balance disk 1 -Standing, without support, with feet shoulder-width apart 2 -Standing, without support, feet together 3 -Standing, without support, with preferential foot on the disk and the other suspended in the air (90°knee flexion) 4 -Standing, without support, with unpreferential foot on the disk and the other suspended in the air (90°knee flexion) 5 -Standing, without support, with preferential foot forward 6 -Standing, without support, with unpreferential foot forward 7 -Standing, without support, with preferential leg raised forward 8 -Standing, without support, with unpreferential leg raised forward 9 -Standing, without support, with preferential leg in hip abduction 10 -Standing, without support, with unpreferential leg in hip abduction 11 -Standing, without support, with feet shoulder-width apart, trunk flexion and arms forward 12 -Standing, without support, feet together, trunk flexion and arms forward 13 -Standing, without support, with feet shoulder-width apart and eyes closed 14 -Standing, without support, feet together and eyes closed children < 16°percentile are at risk for motor difficult and children > 16°percentile have no motor difficult detected. According to the MABC-2 tester manual, each child spends about 30-40 min completing all tasks. [/fig] [fig] Figure 7: Balance beams [/fig] [table] Table 2: Training activities with Wii and the three domains [/table] [table] Table 1: Intervention protocol sequence of activities [/table] [table] Table 3: No-Wii Experimental protocol activities [/table]

Development and psychometric testing of an abridged version of Dundee Ready Educational Environment Measure (DREEM) Background: Dundee Ready Educational Environment Measure (DREEM) is a 50-item tool to assess the educational environment of medical institutions as perceived by the students. This cross-sectional study developed and validated an abridged version of the DREEM-50 with an aim to have a less resource-intensive (time, manpower), yet valid and reliable, version of DREEM-50 while also avoiding respondent fatigue. Methods: A methodology similar to that used in the development of WHO-BREF was adopted to develop the abridged version of DREEM. Medical students (n = 418) from a private teaching hospital in Madurai, India, were divided into two groups. Group I (n = 277) participated in the development of the abridged version. This was performed by domain-wise selection of items that had the highest item-total correlation. Group II (n = 141) participated in the testing of the abridged version for construct validity, internal consistency and test-retest reliability. Confirmatory factor analysis was performed to assess the construct validity of DREEM-12. Results: The abridged version had 12 items (DREEM-12) spread over all five domains in DREEM-50. DREEM-12 explained 77.4% of the variance in DREEM-50 scores. Correlation between total scores of DREEM-50 and DREEM-12 was 0.88 (p < 0.001). Confirmatory factor analysis of DREEM-12 construct was statistically significant (LR test of model vs. saturated p = 0.0006). The internal consistency of DREEM-12 was 0.83. The test-retest reliability of DREEM-12 was 0.595, p < 0.001.Conclusion: DREEM-12 is a valid and reliable tool for use in educational research. Future research using DREEM-12 will establish its validity and reliability across different settings. # Background Education environment refers to the diverse physical locations in which the students learn. The educational environment of an institution has direct and indirect influence(s) upon the knowledge gained by the students including the motivation to learn, personal safety and their well-being. Dundee Ready Educational Environment Measure (DREEM) is one of the tools developed specifically to assess the educational environment of medical institutions as perceived by the students. DREEM has 50 items grouped under five domains namely students' perception of learning (SPoL; n = 12), students' perception of teachers (SpoT; n = 11), students' academic self-perception (SASP; n = 8), students' perception of atmosphere (SPoA; n = 12), and students' social self-perception (SSP; n = 7). It is a generic and culturally non-specific tool which has been translated and validated in at least eight languages and is used worldwide [bib_ref] Development and validation of the Dundee Ready Education Environment Measure (DREEM), Roff [/bib_ref]. In addition to being used to diagnose the deficiencies of the current educational environment, DREEM has also been used to compare different groups, to monitor the same cohort over a period of time and to assess factors influencing the educational environment [bib_ref] DREEM and beyond; studies of the educational environment as a means for..., Whittle [/bib_ref]. DREEM was used among undergraduates [bib_ref] Determining the quality of educational climate across multiple undergraduate teaching sites using..., Varma [/bib_ref] including nursing [bib_ref] Investigating nursing students' perceptions of the changes in a nursing curriculum by..., O&apos;brien A [/bib_ref] and dental students and various post graduate medical students [bib_ref] The potential use of DREEM in assessing the perceived educational environment of..., Jeyashree [/bib_ref] [bib_ref] Evaluation of the learning environment for diploma in family medicine with the..., Khan [/bib_ref] to explore their perceptions of educational environment. Though universally accepted and used, investigators followed their own analysis and reporting system of DREEM data [bib_ref] The Dundee Ready Education Environment Measure (DREEM): a review of its adoption..., Miles [/bib_ref]. Few studies questioned the five-factor structure and construct validity of the original DREEM tool and also tried to develop abridged versions of DREEM [bib_ref] Psychometric properties of DREEM in a sample of Malaysian medical students, Yusoff [/bib_ref] [bib_ref] The Dundee Ready Educational Environment Measure: a confirmatory factor analysis in a..., Yusoff [/bib_ref] [bib_ref] A psychometric appraisal of the DREEM, Hammond [/bib_ref] [bib_ref] The DREEM, part 2: psychometric properties in an osteopathic student population, Vaughan [/bib_ref]. The other studies have developed an abridged version but the properties of the abridged version have not been tested on another cohort. These abridged versions have not been validated nor have they been widely in use. Our study developed and validated an abridged version of the DREEM-50 with an aim to have a less resource-intensive (time, manpower), yet valid and reliable, version of DREEM-50 while also avoiding respondent fatigue. # Methods ## Study design and setting A cross-sectional study was conducted for development of an abridged version of DREEM at a private, tertiary care teaching institute in Madurai, a city in Tamil Nadu state of India. It is one of the two major and the only private institution teaching medicine and allied health sciences in the city. Every year, 150 students are selected through a competitive examination for the undergraduate course in medicine-Bachelor of Medicine and Bachelor of Surgery (M.B, B.S.). ## Study participants A list of the 450 undergraduate medical students currently enrolled in the institute (third, fourth and sixth semester) as on August 2016 was obtained, and all students were invited to participate in the study. These students were divided into two groups group I (students of the fourth and seventh semester, n = 300) and group II (students of third semester, n = 150). ## Data collection The DREEM-50 item version was administered to both groups after obtaining written informed consent, and two separate datasets were maintained. The author was present during the consent process to ensure that clarifications were provided to queries, if any. Students who did not consent and those unavailable after three attempts of contact were excluded from the study. # Statistical analysis Double data entry and validation was done using EpiData version 3.1 (EpiData Association, Odense, Denmark). Items that had to be reverse coded were done so before entry. The data was analysed using STATA (version 12.1, copyright 1985-2011 StataCorp LP USA, serial number: 30120504773). Number and proportion were calculated for sociodemographic characteristics like gender and semester. Mean and standard deviation were calculated for age and for score of each item, domain and total scale of DREEMfull and abridged version. ## Development of an abridged version For the development of the abridged version, the authors followed a procedure similar to that adopted in the development of WHOQOL-BREF, an abridged version of WHOQOL-100, which measures quality of life (QOL) [bib_ref] Development of the World Health Organization WHOQOL-BREF quality of life assessment. The..., The [/bib_ref]. Item-total correlation (r) and domain-total correlation (r) were calculated (group I dataset). Two items with the highest item-total correlation values were selected from each of the five domains of the DREEM questionnaire. When two items had the same correlation value, both were included. Thus, the authors arrived at a 12-item version of DREEM (DREEM-12). The face and content validity of this abridged version was assessed by the three authors, in their capacity as subject experts each with a minimum of 3 years' experience in medical education. ## Validity and reliability of dreem-12 DREEM-50 and DREEM-12 scores were calculated on group II dataset. Internal consistency was measured using Cronbach's alpha. The percentage of variance in DREEM-50 that is explained by the abridged version was calculated using R square. DREEM-12 alone was administered on group II to get the second set of scores of DREEM-12 to check for test-retest reliability (r). Confirmatory factor analysis was performed to assess the construct validity of DREEM-12. A five-factor firstorder model was fit to assess the DREEM-12 structure using the items selected for each DREEM-12 component (items), i.e. SPoT (two), SPoT (three), SASP (three), SPoE (two) and SSP (two). ## Ethics Ethics approval was obtained from institute ethics committee of Velammal medical college hospital and research institute, Madurai, Tamil Nadu. Written informed consent was taken from the students and the consent process was approved by the ethics committee. # Results A total of 277 students from group I participated in the development of the abridged version and 141 students from group II in the validation and reliability of the abridged version. The age and gender distribution of the study population is given in [fig_ref] Table 1: Profile of study participants -undergraduate medical students from Madurai, India in 2016 [/fig_ref]. The item-total correlation and domain-total correlation of the items selected for inclusion in DREEM-12 are given in [fig_ref] Table 2: Domain-wise list of items in DREEM-50 that had the highest item-total and... [/fig_ref]. Correlation between total scores of DREEM-50 and DREEM-12 was 0.88 (p < 0. The item wise and overall scoring system of DREEM-12 was devised in line with the DREEM-50 [fig_ref] Table 3: Items selected to form DREEM-12 and the applicable scoring system [/fig_ref]. When calculated for group II, DREEM-50 and DREEM-12 scores yielded the same interpretations on the students' perceptions about the educational environment: overall and at domain level [fig_ref] Table 4: Comparison of DREEM-50 and DREEM-12 scores and their interpretation calculated for group... [/fig_ref].The test-retest reliability of DREEM-12 was 0.595, p < 0.001. Confirmatory factor analysis of DREEM-12 construct was statistically significant (LR test of model vs. saturated: χ 2 (44) = 80.88, Prob> χ 2 = 0.0006) [fig_ref] Figure 1: Confirmatory factor analysis of DREEM-12 construct [/fig_ref]. # Discussion This study developed and validated DREEM-12, an abridged version of DREEM-50, using two subsets of a population of undergraduate medical students and established good model fit of DREEM-12 by confirmatory factor analysis. Our study, besides developing an abridged version of DREEM-50, has also confirmed its factor structure and established its validity and reliability. We have employed robust data quality management, through double data entry and validation, and statistical techniques to prove the same. Our study has enrolled students from different years of medical education and thus can be considered representative of the perceptions of students in different stages of medical education. The study was limited to only students from a single teaching institution in India. However, ours being a study on educational environment, it is also necessary to point out that the quality standards of a medical college with respect to curriculum, teaching methodology, infrastructure, staffing and assessment methods are strictly governed to be uniform by the Medical Council of India. These standards are uniform for all the colleges countrywide. All colleges, government and private, are to abide by these standards, failing which the recognition of the institute is cancelled and the given institute will no more be able to admit candidates. Hence, it will be safe to say that the educational environment offered in colleges across the country are to be uniform. The students are admitted into the undergraduate medical course by a nationwide common competitive entrance examination-National eligibility entrance test. The students do come from different sociodemographic and cultural backgrounds in different parts of the country. But, we do not expect that to significantly affect their perception of their environment. Thus, while the internal validity of our study is commendable, further multi-centric research is required to Group I-fourth and sixth semester students; group II-third semester students SD standard deviation The psychometric properties of original DREEM-50 were investigated in few studies. Confirmatory factor analysis found that the five-factor structure of DREEM-50 showed poor model fit in studies conducted in Malaysia and Australia [bib_ref] The Dundee Ready Educational Environment Measure: a confirmatory factor analysis in a..., Yusoff [/bib_ref] [bib_ref] The DREEM, part 2: psychometric properties in an osteopathic student population, Vaughan [/bib_ref]. Both these studies came up with an abridged version of DREEM-50 with a good model fit [fig_ref] Table 5: Comparison of items in the abridged version of DREEM developed in our... [/fig_ref]. The Malaysian study derived a 17-item abridged version with good model fit with each domain having three items except the students' perception of atmosphere which had five items. Similarly, the Australian study derived 12-item abridged version with only four domains after removing student's social self-perception. The difference in the items chosen in our study, Malaysian and Australian abridged versions might have been due to the following reasons-the methodology of choosing items into the abridged version was different in the three studies. In our study, we had followed the method used in development of WHO QOL BREF. We included items based on their item-total correlations. While the Malaysian version retained the five-domain structure of the original DREEM, the Australian [fig_ref] Table 3: Items selected to form DREEM-12 and the applicable scoring system [/fig_ref] also figure in the Malaysian 17-item version [bib_ref] Psychometric properties of DREEM in a sample of Malaysian medical students, Yusoff [/bib_ref] [bib_ref] The Dundee Ready Educational Environment Measure: a confirmatory factor analysis in a..., Yusoff [/bib_ref]. None of the items in the Australian abridged version are present in the current DREEM-12 except for item 22 [bib_ref] The DREEM, part 2: psychometric properties in an osteopathic student population, Vaughan [/bib_ref]. Our abridged version is short like the Australian version and maintained the five-domain structure of the original version like the Malaysian version. A study from Ireland also mentioned the poor construct validity of the DREEM-50 and found some 17 items having poor fit indices (< 0.7). This study failed to propose an abridged version (33 items) mentioning two reasons, namely, the weakest items may not be same for different settings and fear of affecting the factor structure [bib_ref] A psychometric appraisal of the DREEM, Hammond [/bib_ref]. Reports on test-retest reliability of DREEM are variable. A study from Brazil reported moderate testretest reliability of 0.43 [bib_ref] Psychometric properties of the Dundee Ready Educational Environment Measure (DREEM) applied to..., Filho [/bib_ref]. Contrarily, a study validating the Greek translation of DREEM, reported 0.90. While a generally accepted cut-off of 0.7 is followed, there are no standards laid for test-retest reliability cut off for instruments measuring psychological parameters [bib_ref] Assessing test-retest reliability of psychological measures, Aldridge [/bib_ref]. # Conclusion DREEM-12, developed as an abridged version of DREEM-50, was found to explain 77.5% of the variance of the scores of the original version and also retained its factor structure, validity and reliability. Future research using DREEM-12 will establish its validity and reliability across different settings. [fig] Figure 1: Confirmatory factor analysis of DREEM-12 construct. SSP-Social Self Perception, ASP-Academic Self perception, SPOT-Students' Perception of Teachers, SPOTe-Students' Perception of Teaching, POE-Perception of Environment three-3, ninet-19, twone-21, fone-41, ffive-45, twtwo-22, ffour-44, two-2, eightn-18, tseven-37, tsix-36, fthree-43. LR test of model vs. saturated: chi2(44)=80.88, Prob > chi2 = 0.0006 [/fig] [table] Table 1: Profile of study participants -undergraduate medical students from Madurai, India in 2016 (n = 418) [/table] [table] Table 2: Domain-wise list of items in DREEM-50 that had the highest item-total and domain-total correlations [/table] [table] Table 3: Items selected to form DREEM-12 and the applicable scoring system [/table] [table] Table 4: Comparison of DREEM-50 and DREEM-12 scores and their interpretation calculated for group II SD standard deviation version had done away with the social self-perception domain. Since our study revealed equally good domain-total correlation for all domains, similar to the Malaysian study, we chose to retain all domains in our abridged version. Eight (items 3, 19, 22, 37, 41, 43, 44 and 45) out of the 12 items selected in our study [/table] [table] Table 5: Comparison of items in the abridged version of DREEM developed in our study with those in Malaysian and Australian versions [/table]

Induced abortion and effecting factors of ever married women in the Southeast Anatolian Project Region, Turkey: a cross sectional study Background: Nearly 10% of the population of Turkey lives in the Southeast Anatolian Project (SEAP) region. The population growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Lifetime induced abortion rate is also high in this region.Public health problems of the SEAP region were investigated in the "SEAP Public Health Project" in 2001 and 2002. As it is one of the most important health problems of the women living in this region; induced abortion was also investigated in this project.Methods: An optimumsample size representing the rural and urban area of the region (n = 1150) was chosen by the State Institute of Statistics by a sampling method proportional to size. 1126 of the area's 1150 houses have been visited and data about induced abortions have been obtained by applying a questionnaire to 1491 ever married women who live in the region.Results: It has been found that 9.0% of these women who had at least one pregnancy in their life had at least one induced abortion. The lifetime induced abortion per 100 pregnancies was found to be 2.45. The primary reason given for induced abortions was "wanting no more children" (64.6%). Lifetime induced abortions were 5.3 times greater with women using a family planning method than women not using family planning methods. Lifetime induced abortions were 4.1 times greater with unemployed women than working women. Most of the women have used private doctors in order to have an induced abortion. Although 32. have not yet begun to use a contraceptive method after their last induced abortion, 43.75% of the women have since started to use an effective contraceptive method. 23.96% of them have begun to use an ineffective contraceptive method. Conclusions: Induced abortion is still an important problem at the SEAP region. The results of the study remind us that unemployed women and women who have more than four children is our target group in the campaign against induced abortions. Most of the women use private doctors in order to have an induced abortion. Thus, priority must be given to educate private gynecologists with respect to induced abortion. After induced abortions, a qualified family planning consultant can be given to women and they can be secured to use a suitable contraceptive method. # Background Abortion is defined by World Health Organization (WHO) as a pregnancy that ends before 28 th week of gestation. Abortions are divided into two groups as 1) induced abortion and 2) spontaneous abortion. The spontaneous abortion rate increases when the maternal and natal care is insufficient. Induced abortions occur at the desire of the couple and an increase in induced abortion rate is a good indicator of insufficient family planning services. The aim of the family planning services is the prevention of unwanted pregnancies. Inadequate access to contraceptive methods, method failure caused by misuse of the methods and non-use of effective methods are the reasons of unwanted pregnancies, which lead women to induced abortion [bib_ref] Contraceptive Technology: International Edition, Hatcher [/bib_ref]. Induced abortions have been used as a family planning method for many years and become an important problem in women's health especially in developing countries. It is one of the main causes of death of women of reproductive age [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. Induced abortions have many health disadvantages especially when performed in unsafe conditions. In a study it has been found out that abortion may be a risk factor for subsequent depression for a period of 8 years after pregnancy occurs [bib_ref] Depression associated with abortion and childbirth: a long-term analysis of the NLSY..., Cougle [/bib_ref]. In another study the mortality rate for induced abortion was found to be 5.3% and this accounted for 21.1% of the total maternal deaths for this period [bib_ref] Morbidity and mortality following induced abortion in Nnewi, Ikechebelu [/bib_ref]. As it is seen from these studies, induced abortions have many health disadvantages for women and thus induced abortions should not be used as a family planning method. In Turkey, the Population Planning Law legalized the provision of safe abortion services within ten weeks in May 1983. As a result, the facility to terminate unwanted pregnancies in safe conditions has been provided [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. But induced abortion rates are different in the different regions of Turkey. Lifetime induced abortion rate is 26.6% for the whole of Turkey. However, this rate differs from 17.8% to 30.9% for the different regions of Turkey [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. Nearly 10% of the population of Turkey lives in the Southeast Anatolian Project (SEAP) region. The popula-tion growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Public health problems of the SEAP region were investigated in the "SEAP Public Health Project" in 2001 and 2002. Induced abortion was one of the health problems investigated in this project. # Methods The Southeast Anatolian Project (SEAP) region has a population of approximately 6 million people and nearly 10% of the population of Turkey lives in this region. The population growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Public health problems of the SEAP region was investigated in the "SEAP Public Health Project" and this project was supported by the SEAP Regional Development Management of Prime Ministry Republic of Turkey and conducted by a consortium constituted by the Turkish Parasitology Association, Gaziantep University, Dicle University (in Diyarbakır province) and Harran University (in Şanlıurfa province). Induced abortion -an important problem for women -was investigated in this project in 2001 and 2002. The population of the nine provinces in the region is 6,128,973. In order to investigate the public health problems of the region such as abortion, an optimumsample size which represents the rural and urban area of the region was determined as 6900 (d = 0.03, p = 0.04, α = 0.01). This number (6900) was divided to the average number of households (approximately 6 people live in each house in the SEAP region) and the number of houses in the sample was found to be 1150. An optimum sample size representing the rural and urban area of the region was chosen by the State Institute of Statistics by a sampling method proportional to size. Questionnaires were prepared by the academic staff of public health departments of medical faculties of the two universities (Gaziantep and Dicle Universities). Three of the questionnaires were for individuals (the questionnaire of 5 year and older girls and women, the questionnaire of 5 year and older boys and men, and the questionnaire of 0-59 month's old children) and one of the questionnaires was about the house conditions. Before the study, the questionnaires were applied to houses that were not in the study sample as a pilot study and then checked. A team for questionnaire application was constituted in every province and the teams were educated about the questionnaires. These teams visited all of the houses in the sample with a public health specialist (the head) and applied the questionnaires by face-to-face interview. Data about the people living in the house were obtained by the house questionnaire. Data about the demographic features of women, fertility, and features about abortion were obtained by the questionnaire from 5 year and older girls and women. Educated nurses (all of them were women) applied the questionnaire to women by face-to-face interview in a separate room. 1126 households of the area's 1150 houses participated to the survey. Households of the 24 houses were not found at home during the study. There were 1491 ever married (married, divorced or widow) women in the 1126 houses that participated in the survey. The data were evaluated using the SPSS 5.0 and Excel programs. Chi-square, Student's t test and logistic regression analysis were used for the statistical analysis. # Results There were 1491 ever married (married, divorced or widow) women in the 1126 houses that participated in the survey. 1266 (84.9%) of these women had at least one pregnancy in their life. 9.0% of the women who were ever married and who had at least one pregnancy in their life have had at least one induced abortion in the past. The rate of the women who have had two or more induced abortions was found to be 3.63%. The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some basic factors are shown in [fig_ref] Table 1: The lifetime induced abortion rates of the women who were ever married... [/fig_ref]. The percentage of women who have made at least one lifetime induced abortion was higher in women living in urban areas (10.39%) than women living in rural areas (6.85%, p < 0.05). The percentage of lifetime induced abortion was higher in 35-49 age group (especially in 45-49 age group) than the other age groups (p < 0.01). Lifetime induced abortion rate was 7.15% in illiterate women and 10.53% in literate women and was higher among women who graduated from a primary school or higher (%12.77, p < 0.05). The percentage of women who have had at least one induced abortion was found to be higher in unemployed women and Turkish women than the other groups (p < 0.0001, [fig_ref] Table 1: The lifetime induced abortion rates of the women who were ever married... [/fig_ref]. The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some fertility characteristics are shown in [fig_ref] Table 2: The lifetime induced abortion rates of the women who were ever married... [/fig_ref]. The age of the women at her first birth, number of still birth and spontaneous abortion did not affect the rate of lifetime induced abortion. The number of living children of the women was related to the number of lifetime induced abortion. The induced abortion rate was significantly high in women having 4 or more children (p < 0.01). A similar relationship was found between induced abortion and total number of pregnancies. Lifetime induced abortion was 12.09% among women who had five and more pregnancies and this was higher than the other groups (p < 0.001). Lifetime induced abortion was found to be significantly higher in women who had got pregnant with their last child without the desire of both of the couples, who wanted no more children and who were using a family planning method (13.31%, 11.95% and 15.21% respectively) [fig_ref] Table 3: The number of lifetime induced abortions of women who were ever married... [/fig_ref]. The lifetime induced abortion rates have been evaluated considering all of the factors thought to be related with induced abortion and has been shown in Tables 1, 2, 3. When we evaluate the results of logistic regression analysis; the number of total pregnancies has been found to be the factor mostly affecting the lifetime induced abortion status [fig_ref] Table 4: The results of logistic regression [/fig_ref]. Every one point increase of the total number of pregnancies increases the risk of making induced abortion by 1.17 times. The family planning method usage status of the women and the employment status of the women were the other two variables affecting the lifetime induced abortion status of the women. The risk of lifetime induced abortion was found to be 5.4 times greater with women using a family planning method than women not using family planning methods. The lifetime induced abortion risk was found to be 4.1 times greater with unemployed women than working women. The rate of induced abortions per 100 lifetime pregnancies -one of the most common indicators of induced abortions -was found to be 2.45. This rate is 1.38 at the rural areas and it rises to 3.33 at the urban areas (p < 0.05) [fig_ref] Table 5: The induced abortion rate per 100 lifetime pregnancies among ever married women [/fig_ref]. "Wanting no more children" is the primary reason given for lifetime induced abortion (64.58%). In 63.54% of the lifetime induced abortions both of the couples have decided to the induced abortion together. Most of the lifetime induced abortions take place at the private doctors' consultant room (46.88%) [fig_ref] Table 6: Some characteristics of the women's last induced abortion [/fig_ref]. After lifetime induced abortion, 32.29% of the women have not yet begun to use a family planning method. 43.75% of them have since started to use effective methods and 23.96% of them have begun to use ineffective methods. The usage of effective methods was higher in urban areas, while the usage of ineffective methods was higher in rural areas. Intra uterine devices (IUD) (52.38%) took the first and condom (26.19%) took the second place among the effective family planning methods. Withdrawal, with a rate of 87%, took the first sequence among the ineffective family planning methods [fig_ref] Table 7: Usage of family planning methods after lifetime induced abortion [/fig_ref]. In the study, lifetime induced abortions carried out by the women were also evaluated. The number of the women who have stated that "they have tried to make an induced abortion by themselves" in the past was 64. 24 of these women were from rural areas and 40 of them were from urban areas. The women who intended to carry out an induced abortion by themselves firstly preferred to use drugs (43.8%). Lifting heavy things (35.4%) took the second place. Women who live in rural areas preferred to lift heavy things (64.3%) while women in urban areas preferred to take drugs (50.0%). # Discussion The percentage of having at least one induced abortion among ever married women who had at least one pregnancy in their life in the SEAP region was 9.0% (lifetime induced abortion rate). Approximately one out of ten ever married women has made at least one induced abortion in their life. Also, 2.45 induced abortion per 100 lifetime pregnancies occurred at the region. When we evaluated the results of the Turkish Demographic and Health Survey 1998; (TDHS 1998) (which is conducted to collect data on subjects such as fertility, infant and child mortality, family planning, and maternal and child health on a representative sample of Turkey through the interviews conducted with women of fertile age) the percentage of lifetime induced abortion among ever married women was reported as 18.2% and induced abortion per 100 pregnancies during the five-year period before the survey was 7.6 for the East Anatolian region (the East Anatolian and the Southeast Anatolian Regions were evaluated together as one region and the SEAP provinces take part in this region). The SEAP rates were lower than the TDHS 1998. In the TDHS 1998 the lifetime induced abortion rates of the East Anatolian Region were given. The Southeast Anatolian region provinces were evaluated in this region. This study was conducted in the Southeast Anatolian Region only. The general features and health conditions of the Southeast Anatolian Region are worse than the East Anatolian Region, explaining why the rate (9%) is lower than the TDHS 1998. There is a decrease in the lifetime induced abortion rate in the course of time compared with the TDHS 1998. Also, there is a decrease in the lifetime induced abortion rate in the same region (in the East Anatolian provinces) when the data of the TDHS 1993 is compared with the data of the TDHS 1998. Induced abortion rate per 100 pregnancies during the five-year period before the survey has decreased to 7.6 from 8.7 in the course of time [bib_ref] Health Ministry General Directorate of Maternal and Child Health and Family Planning..., Dervişoğlu [/bib_ref]. A similar decrease was seen when the Turkey Reproduction Survey-1978 was compared with the TDHS 1998 [bib_ref] A cross sectional study on abortion, Akadli [/bib_ref]. In another study conducted in Turkey; abortion rate (both induced and spontaneous abortions) of ever married women was found to be 14.9% in 1991. In a resent study conducted in Manisa in 2000 induced abortion rate per 100 pregnancies during the five-year period before the survey was found to be 12.1% [bib_ref] Induced Abortions in Manisa, Turkey, Gönül [/bib_ref]. It is seen that the induced abortion rate is decreasing not only in the SEAP region but also in other regions of Turkey in the course of time. In a study conducted by Senlet et al. it is reported that there is a decline in induced abortion rates in Turkey [bib_ref] The role of changes in contraceptive use in the decline of induced..., Senlet [/bib_ref]. However, this low lifetime induced abortion rates do not show a success because unintended pregnancies end with births in the region. As a matter of fact, 30.1% of the latest births of the women during the last five year period were not desired by both of the couples in the Southeast Anatolian region [bib_ref] Attitudes about family planning in the Southeast Anatolian project region, Özçirpici [/bib_ref]. Also, total fertility rate of the women was 4.2 in the East Anatolian region. The high fertility rate and the high rate of ending unintended pregnancies with births is the real cause of the low lifetime induced abortion rate in the region. The rate of induced abortion was higher in urban areas than rural areas. This was similar with the TDHS 1998. Lifetime induced abortion rate was 7.15% among illiterate women, 10.53% among literate women and was higher among women graduated from primary school or higher (% 12.77, p < 0.05). In a study conducted by Akın et al. similar results have been found [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. Education is a very important factor effecting induced abortion rate. In the logistic regression analysis the total number of pregnancies of the women, the family planning method usage status of the women and the employment of the women have been evaluated as the independent factors affecting lifetime induced abortion. As the total number of pregnancies increases, lifetime induced abortion risk increases (odds ratio is 1.7). Women who have more than four children may be the target group of the studies planned on this subject. In a study conducted by Akın et al. a similar odds ratio (1.1) have been found [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. Lifetime induced abortions were 4.1 times greater with unemployed women than working women. This was due to the fact that these women have lower family planning usage rates but their pregnancy rate was high. These results remind us that unemployed women and women who have more than four children must be our target group in the campaign against induced abortions as a family planning method. Lifetime induced abortions were 5.3 times greater with women using a family planning method than women not using family planning methods. I.e. the usage of family planning methods are 5.3 times higher among the women who have had an induced abortion in the past. In a study conducted by Akın et al. similar results were reported during the five-year period before the survey (odds ratio is 2.9) [bib_ref] Induced abortions in Turkey. In In Maternal health and family planning services..., Akın [/bib_ref]. Lifetime induced abortions have usually taken place at a health facility and with the assistance of health personnel. After these lifetime induced abortions, a qualified family planning consultant can be appointed to these women and they can be encouraged to use a suitable contraceptive method. The rate of effective family planning method usage after induced abortion was 43.7% in our study. The same rate was 34.2% in the TDHS 1998 during the five- year period before the survey. There is an increase in the rate of effective family planning method usage after lifetime induced abortion and this increase is pleasing but it is still insufficient. This increase is thought to be one of the reasons of the decrease in induced abortion rates. Similarly, Senlet and et al has reported that one of the reasons of decrease of the induced abortion rates in Turkey is due to this factor [bib_ref] The role of changes in contraceptive use in the decline of induced..., Senlet [/bib_ref]. After lifetime induced abortion, 32.3% of the women were not using a family planning method in the study and this was nearly the same with the percentage evaluated in the TDHS 1998 during the five-year period before the survey (32.1%). There was no important change during the past four years. In another study in Turkey 25% of the women did not begin to use a family planning method after induced abortion. In two other studies conducted in Turkey it has been found out that approximately 20% of the women did not begin to use a family planning method after induced abortion [bib_ref] Contraceptive practices of women after abortion in Turkey, Karabacak [/bib_ref] [bib_ref] The effectiveness of preabortion contraception counselling, Ortayli [/bib_ref]. Also, 23.9% of them have begun to use an ineffective method in our study. These data shows that the family planning services are not adequate at the institutions where induced abortion is performed. Private Doctors (46.88%) and public hospitals (27.08%) were the fist two places where the women applied to have an induced abortion. Similar results have been found in the TDHS 1998 for the Eastern Anatolian provinces during the five-year period before the survey (68.4% and 19.7% respectively). Similar results were obtained in another study in our country and it has been found out that 50% of the induced abortions were made by private doctors and private doctors were the first place chosen for induced abortion [bib_ref] The frequency of abortion and the effecting factors among 15-49 years old..., Şenol [/bib_ref]. Thus, priority must be given to educate private gynecologists. After lifetime induced abortion, 67.71% of the women have begun to use a family planning method in our study. The primary reason given for the last induced abortion was "wanting no more children" (64.5%) and this is similar with the data of the TDHS 1998. This is also another indicator for high unintended pregnancy rates and insufficient family planning services in the region. Similar results have been obtained in a different study in our country. In this study 47.6% of the women requested an induced abortion because they wanted no more children [bib_ref] Reasons why women have induced abortions in a developing country, Uygur [/bib_ref]. Although the rate of lifetime induced abortions are decreasing in the course of time it is still an important health problem in the SEAP region. Unintended pregnancy and total fertility rates of the region is still higher than the other regions of Turkey. Thus, family planning services, the educational level of women and the status of women need improvement. [table] Table 1: The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some basic factors One and two or more induced abortions have been evaluated together in the statistical analyses. [/table] [table] Table 2: The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some fertility characteristics *One and two or more induced abortions have been evaluated together in the statistical analyses. [/table] [table] Table 3: The number of lifetime induced abortions of women who were ever married and who had at least one pregnancy in their life according to some factors related with family planning *One and two or more induced abortions have been evaluated together in the statistical analyses. **The evaluations include the ones who have answered the questions. [/table] [table] Table 4: The results of logistic regression [/table] [table] Table 5: The induced abortion rate per 100 lifetime pregnancies among ever married women [/table] [table] Table 6: Some characteristics of the women's last induced abortion [/table] [table] Table 7: Usage of family planning methods after lifetime induced abortion [/table]

Assessing infant cognition in field settings using eye-tracking: a pilot cohort trial in Sierra Leone Objectives To investigate the feasibility of eyetracking-based testing of the speed of visual orienting in malnourished young children at rural clinics in Sierra Leone. Design Prospective dual cohort study nested in a clusterrandomised trial. Setting 8 sites participating in a cluster-randomised trial of supplementary feeding for moderate acute malnutrition (MAM). Participants For the MAM cohort, all infants aged 7-11 months at the eight sites were enrolled, 138 altogether. For controls, a convenience sample of all non-malnourished infants aged 7-11 months at the same sites were eligible, 60 altogether. A sample of 30 adults at the sites also underwent eye-tracking tests as a further control.Interventions Infants with MAM were provided with supplementary feeding. Outcome measures The primary outcomes were feasibility and reliability of eye-tracking-based testing of saccadic reaction time (SRT). Feasibility was assessed by the percent of successful tests in the infants. Reliability was measured with intraclass correlation coefficients (ICCs). Secondary outcomes were mean SRT based on nutritional state as well as and changes in mean SRT after supplementary feeding of MAM children. Results Infants exhibited consistent orienting to targets on a computer screen (>95% of valid trials). Mean SRTs had moderate stability within visits (ICCs 0.60-0.69) and across the 4-week test-retest interval (0.53) in infants; the adult control group had greater SRT stability (within visit ICC=0.92). MAM infants had a trend toward higher adjusted SRT at baseline (difference=12.4 ms, 95% CI −2 to 26.9, p=0.09) and improvement in SRT 4 weeks thereafter (difference=−14 ms, 95% CI −26.2 to −1.7, p=0.025) compared with age-matched controls. Conclusions The results demonstrate the feasibility of eye-tracking-based testing in a resource-poor field setting and suggest eye-tracking measures have utility in the detection of group level effects of supplementary feeding. # Introduction Children who lack access to appropriate nutrition, care and environmental stimulation in early childhood are at heightened risk for compromised learning and cognition, as well as persistent cognitive, emotional and social problems in adulthood. [bib_ref] Nutrition and brain development in early life, Prado [/bib_ref] [bib_ref] Cognitive recovery in socially deprived young children: the Bucharest early intervention project, Nelson [/bib_ref] [bib_ref] Nutrition and the developing brain: nutrient priorities and measurement, Georgieff [/bib_ref] [bib_ref] Inequality in early childhood: risk and protective factors for early child development, Walker [/bib_ref] [bib_ref] Impaired IQ and academic skills in adults who experienced moderate to severe..., Waber [/bib_ref] [bib_ref] Infant malnutrition predicts conduct problems in adolescents, Galler [/bib_ref] [bib_ref] Malnutrition in the first year of life and personality at age 40, Galler [/bib_ref] Although anthropometry offers a crude assessment of development, it remains the dominant means of assessing undernutrition in its acute and chronic forms. Field-applicable tests of neurocognitive function could add a dimension to the assessment of childhood development and contribute to our understanding of the process and effects of acute and chronic malnutrition. [bib_ref] High-Oleic ready-to-use therapeutic food maintains docosahexaenoic acid status in severe malnutrition, Hsieh [/bib_ref] As a first step toward investigating the acute neurocognitive effects of early childhood undernutrition, we examined the feasibility and measurement properties of visual orienting testing in infants in low-resource settings. The capacity to orient eyes to visual objects (eg, abrupt onset of a stimulus) and hold them on key sources of information (eg, faces) emerges rapidly during the first 6 months of life in infants, [bib_ref] Early-onset binocularity in preterm infants reveals experience-dependent visual development in humans, Jandó [/bib_ref] [bib_ref] Measuring the development of social attention using Free-Viewing, Frank [/bib_ref] [bib_ref] Seeing faces is necessary for face-domain formation, Arcaro [/bib_ref] [bib_ref] Associations between the developmental trajectories of visual scanning and disengagement of attention..., Hunnius [/bib_ref] [bib_ref] Automatic detection of attention shifts in infancy: eye tracking in the fixation..., Kulke [/bib_ref] [bib_ref] Age-related change and stability of individual differences in infant saccade reaction time, Canfield [/bib_ref] and provides a foundation for the child's ability to interact with the physical and social environment. [bib_ref] The building blocks of cognition, Rose [/bib_ref] No studies to date have tested whether measures of visual orienting are sensitive to short-term changes in neurocognitive status in individual children, but there is evidence of this utility in adults: the mean latency of visual orienting changes in response to mild Strengths and limitations of this study ► Extension of technologically advanced eye-tracking testing into underserved and severely resourcelimited settings. ► Robust testing of the reliability of the method employed. ► Repeated measurements collected for infants. ► Inclusion of two control groups, age-matched infants as well as adults, to investigate sources of variability in test results. ► Cohorts are small and included infants from a single district in rural Sierra Leone. Open access traumatic brain injuries (concussion) and provides a reliable measure for monitoring neurocognitive recovery in individual persons over a few days' time. [bib_ref] Saccadometry: the possible application of latency distribution measurement for monitoring concussion, Pearson [/bib_ref] Studies of visual orienting in infants have shown positive associations between the speed of visual orienting at 4-7 months of age and performance IQ at 4 years of age, [bib_ref] Infant expectations and reaction time as predictors of childhood speed of processing..., Dougherty [/bib_ref] as well as executive function at age 11. [bib_ref] Implications of infant cognition for executive functions at age 11, Rose [/bib_ref] Reduced orienting to faces in infancy has been associated with a heightened risk for autism [bib_ref] Attention to eyes is present but in decline in 2-6-month-old infants later..., Jones [/bib_ref] and propensity to social-behavioural problems at 4 years. [bib_ref] Infants' attention bias to faces as an early marker of social development, Peltola [/bib_ref] Visual orienting testing in infants has benefited from the replacement of manual methods of eye movement coding with more automated methods of eye-tracking that use remote infrared light sources and cameras. The majority of studies examining infant visual orienting by automated eye-tracking come from high-resource laboratories in Western countries, but similar research has recently been expanded to assess visual orienting [bib_ref] Eye-tracking-based assessment of cognitive function in low-resource settings, Forssman [/bib_ref] [bib_ref] Associations between individual variations in visual attention at 9 months and behavioral..., Pyykkö [/bib_ref] [bib_ref] Implementing neuroimaging and eye tracking methods to assess neurocognitive development of young..., Katus [/bib_ref] [bib_ref] Early development of visual attention in infants in rural Malawi, Pyykkö [/bib_ref] as well as other aspects of cognition in children in less studied populations in mid-income and low-income countries. [bib_ref] The feasibility of automated eye tracking with the early childhood vigilance test..., Boivin [/bib_ref] [bib_ref] The feasibility of an automated eye-tracking-modified Fagan test of memory for human..., Chhaya [/bib_ref] [bib_ref] Infant gaze following depends on communicative signals: an eye-tracking study of 5-to..., Hernik [/bib_ref] These studies have shown that eyetracking can be performed in low-resource settings, typically at central healthcare facilities, [bib_ref] Early development of visual attention in infants in rural Malawi, Pyykkö [/bib_ref] [bib_ref] The feasibility of automated eye tracking with the early childhood vigilance test..., Boivin [/bib_ref] [bib_ref] The feasibility of an automated eye-tracking-modified Fagan test of memory for human..., Chhaya [/bib_ref] although the use of a portable system at participants' homes in rural Vanuatu has also been reported. [bib_ref] Infant gaze following depends on communicative signals: an eye-tracking study of 5-to..., Hernik [/bib_ref] A study conducted in Malawi found no association between eye-tracking measures of visual orienting at 7-9 months and tests of cognitive and motor function at 18 months. [bib_ref] Early development of visual attention in infants in rural Malawi, Pyykkö [/bib_ref] However, positive correlations were found between eye-tracking measures of novelty preference (ie, preferential looking at a novel vs previously shown face) and conventional tests of cognitive function in 6-12 month-old Ugandan children. [bib_ref] The feasibility of an automated eye-tracking-modified Fagan test of memory for human..., Chhaya [/bib_ref] The psychometric properties of eye-tracking measures (ie, reliability) are critical to assess the utility of this method in monitoring short-term cognitive changes in children. To our knowledge, only one of the studies conducted in low-resource settings used repeated measurements to assess reliability, showing low correlation (0.28) for the mean latency of visual orienting over a 2-month testretest interval. [bib_ref] Early development of visual attention in infants in rural Malawi, Pyykkö [/bib_ref] It is possible, however, that the reliability of the measurements was degraded by the low sampling rate of the eye tracker used (60 Hz) as well as the low number of valid test trials [bib_ref] Inequality in early childhood: risk and protective factors for early child development, Walker [/bib_ref] [bib_ref] Impaired IQ and academic skills in adults who experienced moderate to severe..., Waber [/bib_ref] [bib_ref] Infant malnutrition predicts conduct problems in adolescents, Galler [/bib_ref] [bib_ref] Malnutrition in the first year of life and personality at age 40, Galler [/bib_ref] [bib_ref] High-Oleic ready-to-use therapeutic food maintains docosahexaenoic acid status in severe malnutrition, Hsieh [/bib_ref] per participant. Direct comparisons of groups of malnourished and non-malnourished children on eye-tracking measures are lacking. Weight-for-age z-scores were not correlated with the latency of visual orienting in 9-month-old Malawian infants, but the association may have been affected by the low reliability of the eye-tracking measurements. A recent study conducted in rural Malawi showed no effects of a complementary feeding intervention (one egg per day) on the latency of visual orienting or novelty preference in 12-15 month-old Malawian children. [bib_ref] Early child development outcomes of a randomized trial providing 1 egg per..., Prado [/bib_ref] However, a study in the USA reported faster mean saccadic reaction times (SRTs) at 4-13 months of age in infants whose mothers were supplemented with 930 versus 480 mg of choline per day during the third trimester of pregnancy. [bib_ref] Maternal choline supplementation during the third trimester of pregnancy improves infant information..., Caudill [/bib_ref] We expanded previous eye-tracking studies in lowresource settings by examining whether mean SRTs provide a reliable measure of cognitive changes in acute malnutrition in infants living in villages in rural, Sub-Saharan Africa, where poor nutrition and growth failures are common.Mean SRT has been found to have moderate test-retest reliability in studies in high-resource settings (r=0.65). [bib_ref] Evaluation of psychometric properties of the gap-overlap task in 10-month-old infants, Cousijn [/bib_ref] Our aims were threefold. First, we examined the feasibility and measurement properties of SRT testing in infants in resource-poor settings by using a battery-powered eye tracker in a designated tent. Second, we tested the hypothesis that measures of visual orienting are associated with the nutritional status of the child, as demonstrated by slower SRT in malnourished children as compared with non-malnourished controls. Third, we examined whether visual orienting benefits from nutritional intervention. # Methods ## Patient and public involvement Community leaders and local healthcare workers were engaged prior to the study to discuss the purpose of the study and nature of participation. Village leaders were also involved in site selection. Regular, open progress meetings with village leaders and local healthcare workers were held semiannually. A meeting for dissemination of results was held in August 2019 in Pujehun District. ## Participant recruitment and design Data for children were collected as a part of a larger intervention study comparing the cost-effectiveness of four supplementary foods in the treatment of moderate acute malnutrition (MAM) in Pujehun District, Sierra Leone, while data for adults were collected as part of a study assessing treatment approaches for MAM based on severity of wasting (figure 1). [bib_ref] Effectiveness and costeffectiveness of 4 supplementary foods for treating moderate acute malnutrition:..., Griswold [/bib_ref] In the study setting, 29.9% of the population falls in the lowest quintile for income and 27.3% in the second lowest. The four foods intervention study was implemented in 29 Peripheral Health Units (PHUs), which were randomised to administer one of four supplementary foods to children 6-59 months of age diagnosed with MAM. Inclusion criteria were diagnosis of MAM, defined as mid-upper arm circumference (MUAC) ≥11.5 cm and <12.5 cm without bipedal oedema, caregiver willingness to return to clinic every 2 weeks for assessment and ability of the child to consume the supplementary food as demonstrated at enrolment. Exclusion criteria included developmental disability, congenital malformation and current or recent (up to 1 month) enrolment in a separate supplementary feeding programme. Children were evaluated by anthropometry every 2 weeks, and for a minimum of 2 weeks, until a programmatic outcome was reached. These included recovery, development of severe acute malnutrition, death, loss to follow-up or not recovered despite 12 weeks of supplemental feeding. They received one of the following foods: (1) super cereal plus with amylase (SC+A); (2) corn-soy blend plus and fortified vegetable oil (CSB+ w/oil); (3) corn-soy whey blend and fortified vegetable oil (CSWB w/oil) and (4) ready-to-use-supplementary food (RUSF) (online supplemental table 1). These foods were not formulated to improve cognition or visual function, but rather for nutritional recovery from MAM. All infants 7-11 months of age with MAM who were enrolled into the parent study over a 10-month period at the largest 8 of the 29 PHUs were also enrolled into this dual cohort substudy. The two largest PHUs for each study arm were chosen to participate in the substudy. Control children were recruited at these same 8 PHUs and over the same period of time. Controls were recruited sequentially and assessed via the same inclusion criteria previously listed, with the exception of having an MUAC ≥12.5 cm. Beyond the exclusion criteria of the parent study, three additional criteria were applied to both groups: (a) a known history of visual problems; (b) any neurological problem; (c) inadequate number of valid tests (ie, <10 valid SRTs/visit). In addition, controls were excluded if they developed malnutrition by the second clinic visit. A target sample size of 30 adults aged 18-40 from these same 8 PHUs were also recruited and enrolled sequentially as a convenience sample. Exclusion criteria were (a) a known history of visual problems; (b) any neurological problem; (c) inadequate number of valid tests (ie, <10 valid SRTs/visit). All participants enrolled in the study were assessed by eye-tracking-based tests of visual orienting and by conventional observational tests of oculomotor function. Children were tested at enrolment and again 4 weeks later. The 4-week time period was chosen because all MAM children received 4 weeks of rations, even if they achieved recovery after 2 weeks. The four foods MAM study received ethical approval from the Tufts University Health Sciences Institutional Review Board, the Washington University in St Louis School of Medicine Institutional Review Board and the Sierra Leone Ethics and Scientific Review Committee, while the MAM study in which adult controls were recruited received ethical approval from the Washington University and Sierra Leone review boards. The project was introduced to communities through an open meeting with the chiefs and village leaders. After all questions and comments were addressed, agreement was obtained for offering the study in their community. Following this, open community meetings were held detailing the study, usually attended by 25-50 women, presumably mothers of the beneficiaries. Informed consent was obtained from all caregivers of participants with a private oral explanation of the procedure, risks and benefits. All beneficiary caregivers were illiterate, and signed or thumb-printed the consent form on behalf of their infants. The trial was registered at Clinicaltrials. gov (NCT03146897). ## Open access Eye-tracking testing Setting and equipment Infants were assessed in a 1.5 m (width)×1.5 m (length)×2 m (height) canopy tent with custom-made walls of monochromatic dimout fabric (figure 2). The tent was mounted in the vicinity of a PHU, typically on a covered outdoor porch area. The caregiver held the infant in a baby carrier so that the infant's eyes were facing forward and positioned at approximately 60 cm viewing distance from a 19-Inch Acer V196L LCD monitor (resolution: 1280×1024, refresh rate: 60 Hz, response time: 5 ms, manufacturer: Acer, San Jose, California, USA) and a screen-based Tobii X3-120 eye tracker (Tobii Technology, Stockholm, Sweden). The monitor and the eye tracker were placed on a wooden desk that was custom-made to be at suitable height for the participants in the sample age range. The infants were monitored during testing via a video recorder and a baby monitor. The video recorder (Sony HDR-CX240E, Sony Electronics, San Diego, California, USA) was mounted on top of the monitor. The baby monitor (Summer Infant, Woonsocket, Rhode Island, USA) was placed next to the monitor on the desk. During the assessment, infants were presented with short sequences of visual and audiovisual stimuli on the screen while their point of gaze in the display area was tracked (120 samples/s). The presentation of the stimuli on the screen and the synchronisation of the stimulus event data and eye-tracking data were controlled by a Python script (https://github.com/infant-cognition-tampere/drop) using Psychopy functions [bib_ref] PsychoPy-Psychophysics software in Python, Peirce [/bib_ref] and a Tobii SDK plug-in. The script includes a graphical user interface that allowed the data collector to follow real-time visualisations of the stimulus presentation and eye-tracking data streams throughout the testing (figure 2). The software was run on Linux operating system (Mint 17) and a Lenovo X260 laptop computer (Lenovo, Morrisville, North Carolina, USA), and was connected to the stimulus display via an HDMI-DVI cable and to the eye tracker via an Ethernet cable and a TRENDnet 4-Port Broadband Router (TRENDnet, Torrance, California, USA). The devices used in the testing were powered by a solar-chargeable battery (GoalZero Yeti 400, NRG Energy, Houston, Texas, USA). A fully charged battery supplied power for a full day of testing. ## Procedure and stimuli A Sierra Leonian Mende-speaking data collector was trained to perform the eye-tracking assessments. The data collector seated the caregiver holding the infant in a forward-facing baby carrier such that the infant's eyes were at the optimal distance from the eye tracker. Small variation in sitting height was adjusted for by using pillows. The data collector instructed the caregiver to turn their head and eyes to the side (~90° from the screen) and to avoid looking at the screen during the assessment. The data collector then moved outside the tent laboratory for the duration of testing while constantly monitoring the caregiver and the infant through two monitors. One monitor showed an overall view of the caregiver and the infant. The other monitor showed a visualisation of infant gaze position on the screen. The test consisted of two sessions that lasted 3-4 min each. These sessions were composed of an alternating sequence of dynamic stimuli, including (1) calibration targets, (2) videos depicting a short (5 s) or a longer (9-15 s) dyadic social scene with faces and (3) saccade targets. A design in which calibration targets, naturalistic videos of social scenes and saccade targets were presented in an alternating sequence was used to minimise the monotony of the test sessions. The order of the stimuli was the same for all infants: (1) 3-9 calibration targets, (2) a long social scene (1), (3) five saccade targets, (4) a short social scene (1), (5) five saccade targets, (6) a long social scene (1), (7) five saccade targets, (8) a short social scene (1) and (9) five saccade targets. Infants completing the required two sessions saw a total of 6-18 calibration targets, eight blocks of five saccade targets (40 saccade targets in total) and four long and four short social scenes on each visit. Further details of the stimuli are provided below. While the infants were viewing the stimuli on the screen, their pupil diameter, point of gaze in the display area and position of the eye in a three-dimensional user Open access coordinate system were recorded (120 samples/s). If the infant became restless, inattentive or fussy during the procedure, or if the eye-tracking system lost contact with the eyes, the experimenter paused the test and entered the laboratory to administer a break and/or to perform required adjustments (eg, adjusted the infant's position). ## Calibration targets Calibration targets were short sequences of stimuli which started by the appearance of an audiovisual animation (5.7°×5.7°) in one of three locations: the centre (0°, 0°), bottom-left (−11.4°, −11.4°) or top-right (11.4°, 11.4°) corners of the display area. After the infant looked at the animation or a 3-s timeout value passed, the animation was replaced by an isoluminant white disc (1.3°×1.3°) for one second, after which the original animation reappeared in the same location and was played for another second. The targets were presented up to two times if less than 75% of the recorded point of gaze samples were valid for more than one of the targets. Calibration of the raw point-of-gaze estimates was performed using a similarity transformation estimator (https://pypi.org/project/ nudged/) on samples that fell within a 9°×9° rectangle surrounding the white discs. The accuracy of the calibrated point-of-gaze estimates was verified by calculating the Euclidean distance of the corrected estimates from the location of the targets at the start of the experiment and half-way through the experiment. Similar comparisons were performed for the raw, uncalibrated point of gaze estimates, and the method that provided the lowest error was used: corrected values were superior for 88% of infants for visit 1 data and 83% for visit 2 data. ## Saccade targets Saccade targets were colourful cartoon animations of common objects (eg, fish, face, mouse, pig, soccer ball or bird), subtending a 5.7°×5.7° visual angle. Following a previous study using a similar approach, 36 the targets were presented one at a time without onset delay so that the first target was always presented in the centre of the screen (0°, 0°), and each subsequent target in a new randomly chosen on-screen location 10° away. After appearing on screen, the animations remained paused at the first frame and the accompanying audio silenced until the infant looked at the animation (ie, the first gaze point overlapping with the target area was recorded) or a 1-s wait period elapsed. The animation was subsequently played for 2500 ms with an accompanying sound (eg, water bubbling, bird singing or animal sounds, or non-linguistic utterances). The initial stimulus was not included in analysis, as the starting position of the first saccade was unknown. Following the initial stimulus, infants saw a total of five saccade targets in one block, and a total of eight blocks on each visit. Clearly discernible, colourful animations that varied with each presentation were chosen based on infants' known proclivity for novelty and in line with evidence showing superior response rates for this stimulus style. [bib_ref] Effect of stimulus type on the eye movements of children, Irving [/bib_ref] The stimuli were presented in blocks of five targets in the same order for all participants, but the order of the stimuli was randomised within each block. Additionally, the position of the stimulus was randomly chosen from one of two randomly ordered lists. ## Social scenes Videos depicting short social scenes were 5-15 s video recordings of two Sierra Leonian Mende-speaking female models. Two of the videos were short 5 s recordings of two different female models who looked directly at the camera, and thus the infant, and greeted the child in the child's natural language. The other four videos were 9.5-15 s video recordings of four different models again looking at the camera while enacting a short story or a song appropriate for the participants' age range. The videos were taken in various environments (eg, a kitchen, yard or living room area). Infants saw a total of four short videos (two repetitions per video) and four long videos (one presentation per video) on each visit. The videos were similar to those used in previous studies examining infants' fixations to faces. [bib_ref] Attention to eyes is present but in decline in 2-6-month-old infants later..., Jones [/bib_ref] ## Measures The current analyses focused on SRTs. Analyses of the eyetracking data collected during social scene viewing will be reported separately. SRTs in infants have been variably analysed by using a fixed threshold for eye-movement velocity to detect the onset of a saccadic eye movement, [bib_ref] Development and learning of saccadic eye movements in 7-to 42-month-old children, Alahyane [/bib_ref] by parsing the data into periods of saccades and fixations and detecting the latency of the first fixation in the target area, [bib_ref] Evaluation of psychometric properties of the gap-overlap task in 10-month-old infants, Cousijn [/bib_ref] and by detecting the first recorded point of gaze value in the target area. [bib_ref] Widely applicable MATLAB routines for automated analysis of saccadic reaction times, Leppänen [/bib_ref] For the current study, the method based on the detection of the first recorded point of gaze in the target area was used based on prior work validating this approach for automated SRT analysis in children. [bib_ref] Widely applicable MATLAB routines for automated analysis of saccadic reaction times, Leppänen [/bib_ref] We defined SRTs as the time interval from the onset of the saccade target to the first entry of the gaze into the area of the saccade target. Following the previous validation study using this approach, SRTs were extracted from data that had been filtered with a 15-sample (128 ms) median filter to remove abrupt spikes (figure 3A), and in which the XY-coordinates for the two eyes had been merged by averaging (if both eyes returned a valid data point), or by using the data of one valid eye. Also, a narrow margin (0.9°) was added to the area of the target to accommodate small calibration errors (online supplemental video 1). [bib_ref] Widely applicable MATLAB routines for automated analysis of saccadic reaction times, Leppänen [/bib_ref] The extracted SRTs were regarded as valid and retained in the analyses if the following further constraints were met: (1) the starting position of the saccade was controlled so that the gaze moved from the area of the previous saccade target to the area of the new target (ie, the recorded point of gaze was within the area of the previous target for the majority of the period preceding the saccade, excluding a 50-ms transition period), (2) the analysis period did not have consecutive missing samples exceeding 100 ms, (3) a missing sample did not precede the gaze entry to the target area (ie, the exact Open access point of entry was known within the limits of the eyetracking sampling frequency), (4) the SRT fell inside the commonly used expected time window starting 100 ms after target onset and ending 1000 ms after target onset. SRTs identified as outliers (2.5 SD from mean SRT) were excluded from statistical analyses, which excluded an additional 1.15% of all SRTs. These constraints were similar to those used in the previous validation study, [bib_ref] Widely applicable MATLAB routines for automated analysis of saccadic reaction times, Leppänen [/bib_ref] although some parameter values were adjusted to achieve better control the starting position of the saccades and to accommodate the denser spacing of the stimuli in the current study. Further description of these adjustments and results obtained by the previously used parameters is provided in online supplemental methods and analysis and table S2. [bib_ref] Widely applicable MATLAB routines for automated analysis of saccadic reaction times, Leppänen [/bib_ref] Conventional eye-movement testing Conventional tests included fixation, saccades and smooth pursuit. [bib_ref] Ocular motor score (OMS): a clinical tool to evaluating ocular motor functions..., Olsson [/bib_ref] In the tests of visual fixation, a target (toy) was presented for 4 s in an alternating sequence in the child's frontal, lower left, upper left, lower right and upper right visual field. The child's response (fixation) was scored by using a 3-point scale where 0 indicated no fixation, 0.5 partial and 1 complete fixation of the target. In the saccade and smooth pursuit tests, the first target (a colourful rattle) was presented in the child's field of vision at eye level, centred with respect to the child's line of gaze and at approximately 50 cm from the child's eyes. When the child fixated on the target, a second similar target was presented to the child's lateral field, at the same level as the first target and approximately 50 cm to ## Open access the left or right of the first target. After the child shifted gaze from the first (central) to the second (lateral) target, the lateral target was moved toward the central target. The procedure was repeated twice for both directions. Saccades from the central to the lateral target and smooth pursuit eye movements following the moving target were scored by using a 3-point scale, where 0 indicated absence of saccade or smooth pursuit, 0.5 partial and 1 complete saccade or smooth pursuit. For statistical analysis, the data from the oculomotor testing were aggregated within tasks and subsequently averaged across tasks to a composite measure of oculomotor function. ## Adult control testing The stability of the eye-tracking measures in infants may be affected by 'instrument noise' (ie, limitations of the employed test and analysis setup in reliably detecting and quantifying the timing of saccadic eye movements) and/or more genuine trial-by-trial variability in the actual behavioural responses of infants. [bib_ref] Instrumental and test-retest reliability of saccadic measures, Klein [/bib_ref] To examine major sources of instrument noise in the current setup, we tested a reference group of adult observers with the same eye-tracking setting methodology. Mean SRTs are known to have high within session stability in adults, with Spearman rank correlation for split-half subsets of the data ranging from 0.85 to 0.95. [bib_ref] Individual differences in human eye movements: an oculomotor signature?, Bargary [/bib_ref] The data were analysed using the same pre-processing scripts and parameters as those used with infants. Anthropometric and survey procedures Anthropometry in infants was measured every 2 weeks. Nude weight was measured to the nearest 5 g, recumbent length was measured using a rigid length board in duplicate to the nearest 0.1 cm, and MUAC was measured using a standard insertion tape applied to the left arm and to the nearest 0.1 cm. A socioeconomic survey was conducted with each caretaker. Anthropometric and socioeconomic data were collected on standardised clinic cards and entered into a password protected KoBoCollect database. Anthropometric indices were calculated using WHO Child Growth Standards (Anthro V.3.1, WHO, Geneva). ## Statistical testing For baseline characteristics in the two cohorts, categorical variables were summarised with % and compared using χ 2 , while continuous variables were assessed first for conformance to normality by observing their distribution. If normality was approximated, values were compared using Student's t-test; otherwise, values were compared using Mann-Whitney U test. P values <0.05 were classified as statistically significant. The feasibility and measurement properties of eyetracking tests were examined by calculating descriptive statistics for calibration error, number of valid test trials available for the estimation of mean SRTs and the stability of SRTs. To examine the stability of SRTs (ie, agreement of measures across separate tests), we calculated intraclass correlation coefficients (ICCs) with a two-way random effects model for (1) means computed by dividing the data from visit 1 into two subsets based on odd and even numbered trials and (2) means from visits 1 and 2 (using data from the non-MAM control group). We also calculated the SD of the differences in the measures between visits 1 and 2 (SD Difference ), as well as an estimate of the smallest detectable difference in SRTs for individual infants (±1.96* SD Difference ). Change in SRT was calculated as week 4 SRT minus baseline SRT. Baseline SRT and change in SRT were compared between the two cohorts using independent samples t-test as well as ordinary least squares (OLS) regression with adjustment for age and sex. Change in SRT was also compared by supplement received using both independent samples t-test and OLS regression adjusted for age and sex. Analyses were performed using R (V.4.0.3) and SPSS (V.26.0, IBM, Armonk, New York, USA). # Results ## Sample description Of the 198 infants enrolled in the substudy, 149 participated in the eye-tracking assessments at visit 1 (baseline) ## Open access and visit 2 (4 weeks post-enrolment); 11 did not participate in visit 2 testing and thus were excluded (figure 1). Of those who underwent testing, 3 infants (2%) were excluded due to discovered non-eligibility (neurological history), and 38 infants (19%) were excluded for insufficient number of valid eye-tracking test trials from visits 1 or 2 (<10 trials/visit, [fig_ref] Figure 1: Trial flow diagram [/fig_ref]. Four infants (7%) in the control group developed MAM by visit 2 and were thus excluded. Baseline characteristics of the 142 children show significant differences across all anthropometric criteria between MAM and control children [fig_ref] Table 1: Baseline characteristics of study infants [/fig_ref]. Thirty participants were enrolled in the adult control group and tested with the same paradigm as that used with infants. Twenty-five were retained in the analyses: 17 (68%) were women, with a mean age of 22 (range 18-31). One adult was excluded due to discovered non-eligibility (age >40), three for lost screening or eye-tracking data and one for <10 valid test trials. ## Eye-tracking tests ## Measurement properties In both MAM and control infants, the average number of interruptions per session was <1. Descriptive statistics for calibration error, number of valid trials, probability of response to saccade targets and the within-session and between-session stability of the mean SRTs are provided in table 2. Mean calibration error in the estimates of the point of gaze (ie, distance from the returned value to the actual location of a calibration target) ranged from 0.68° to 1.14° (online supplemental [fig_ref] Figure 1: Trial flow diagram [/fig_ref] , with no significant differences between the MAM and non-MAM control groups. MAM children had more valid trials at baseline than non-MAM controls (p=0.03), but there were no differences between the groups in the postenrolment assessment. Mean SRTs had moderate stability within visits (ICCs 0.60-0.69) and across the 4-week test-retest interval (0.53, figure 3B-D). Using data from the test-retest comparisons in the control group with no predicted change in the behaviour, the estimated SE of SRT means was 25 ms, and the smallest detectable change in individual subject SRT 68 ms. In the adult control group, mean calibration error in the estimates of the point of gaze was 0.9° (max=2.1°) and 1.14° (max=3.3°) horizontally and vertically, respectively. Adults contributed on average 32 valid trials for the analysis of mean SRTs (range 11-39). The rank order correlation of the SRTs for split-half subsets was high (Spearman r=0.87), and the absolute agreement of the two mean SRTs was also high (ICC=0.92, 95% CI 0.83 to 0.97, [fig_ref] Figure 3: A [/fig_ref]. ## Association with nutritional status The MAM group displayed a trend to higher unadjusted and adjusted baseline SRT compared with controls [fig_ref] Table 3: Differences in baseline SRT and change in SRT in children with MAM... [/fig_ref]. SRT shortened in the MAM group after 4 weeks compared with controls [fig_ref] Table 3: Differences in baseline SRT and change in SRT in children with MAM... [/fig_ref]. Changes in SRTs for individual participants are shown in [fig_ref] Figure 4: Mean saccadic reaction times [/fig_ref]. Comparison of change in SRT in infants by supplementary food group revealed a shortening of SRT in each food group compared with change in control, with the exception of CSWB [fig_ref] Table 3: Differences in baseline SRT and change in SRT in children with MAM... [/fig_ref]. ## Conventional oculomotor assessment Complete data on the conventional oculomotor assessment were available for 87 MAM children and 41 controls at visit 1 and 63 MAM and 24 controls at visit 2. Complete data from both visits were available for 57 MAM children and 19 controls. Distribution of responses to individual observational tests assessing saccades, fixations and smooth pursuit of colourful toy objects in a naturalistic interaction situation are shown in online supplemental figure 2. The mean Open access total scores did not differ between the MAM and control groups at baseline [fig_ref] Table 4: Aggregate scores in the coventional tests of oculomotor function in children with... [/fig_ref]. There were also no differences in changes scores between the MAM children and the controls. # Discussion Eye-tracking technologies have been widely used to examine infant attention and cognitive functions in high-resource laboratory settings in Western countries. Recently, the use of this method has also been expanded to low-income countries. This report documents a novel use of eye-tracking in the assessment of neurocognition of infants classified as moderately wasted (experiencing MAM) before and after supplementary feeding in settings that have very few facilities and thus represent the circumstances of many resource-limited communities in Sub-Saharan Africa. The key metric of visual orienting (mean SRT) was obtained for most infants at the first attempt of testing at two time points. Mean SRTs showed moderate levels of within-observer consistency. Relatively high data and participant attrition rates and the presence of withinsubject variability in performance metrics are common findings in studies of infant cognition. The observed levels of data attrition and absolute agreement of mean SRTs within sessions and over a 4-week test-retest interval were within the range of the values reported in previous studies in controlled laboratory environments in infants (r=0.53-0.65). [bib_ref] Evaluation of psychometric properties of the gap-overlap task in 10-month-old infants, Cousijn [/bib_ref] The high precision of the estimates in the adult control group suggests that the comparatively lower stability seen in infants is not due to instrument noise, but rather reflects inherent variability in infant behaviour. Together, these results suggest that there are no major barriers for wider use of eye-tracking in the field in underserved settings. However, the fact that the measures had only moderate within-subject stability in infants indicates that, as currently implemented, eye-tracking measures of visual orienting have more utility in the detection of group level differences (eg, intervention effects) than in the monitoring of neurocognitive changes and recovery in individual infants. Our data revealed a trend toward longer SRT in infants with acute malnutrition, though the 95% CI included negligible (2 ms shorter) and much larger (27 ms longer) differences. This result may reflect that acute malnutrition is but one of many factors that impact cognition, as all participants were from environments that are disadvantaged by many measures. The mean SRTs in the current study were clearly slower than the mean SRTs typically found for infants in this age range. Direct comparisons are complicated by procedural differences between studies, but the potential difference is large: mean SRTs were 400-410 ms in the current study and a recently published study in rural Malawi, [bib_ref] Early child development outcomes of a randomized trial providing 1 egg per..., Prado [/bib_ref] whereas the corresponding values in previous studies in high-resource settings have ranged from 270 to 375 ms. [bib_ref] Automatic detection of attention shifts in infancy: eye tracking in the fixation..., Kulke [/bib_ref] Further research is thus needed to determine the degree and significance of the effects of both acute and chronic malnutrition as well as other poverty-related risk factors on neurocognitive development. As a preliminary demonstration of the potential neurocognitive benefits of supplementary feeding in MAM, our results showed a significant improvement in SRT in the MAM group after 4 weeks of supplementary feeding. This change in SRT was, however, small ## Open access and only evident at the group level. Changes in SRTs showed high variability in individual infants in both the MAM and control groups. The interpretation of the group-level change as indication of improvements in neurocognitive function is supported by previous data showing that specific nutritional supplements result in similar changes in SRT in high resource settings, [bib_ref] Maternal choline supplementation during the third trimester of pregnancy improves infant information..., Caudill [/bib_ref] by the established status of faster orienting as a predictive marker of cognitive function, and, lastly, by the apparent functional benefits that come from faster visual orienting. There were also no indications in the data that the differential change in SRT between the MAM and control groups reflected secondary effects arising from the targeted behaviours becoming more 'measurable' between the two visits (eg, the number of valid trials being disproportionally low in the MAM group on the first visit). Small sample size limited the ability to assess differential effects of the four food groups from the parent study, but the results showed a consistent effect of improvement in SRT, with the exception of CSWB. It is unclear why children receiving CSWB did not demonstrate the same effect, though it is notable that these foods were not designed for neurocognitive benefit, but rather for anthropometric growth. Despite this, the possibility remains that the size of the improvements in the MAM group may change by supplement type, and in, particular, by the use of more neurocognitively targeted nutrients. Our study has multiple limitations. First, it is a cohort study with age-matched controls chosen as a convenience sample and so remains open to confounding factors impacting the results and interpretation. Second, the saccade reaction time methodology requires a minimum number of successful tests, which led to the exclusion of approximately 25% of the initial sample. It is possible these children systematically differed from those included and thus might have altered results. Third, we used different saccade targets on every trial to maximise the interest value of the stimuli. A trade-off of this decision was that some of the variability in SRTs may have resulted from the varying physical and semantic properties of the stimuli. Fourth, while an improvement in SRT was found at group level, the presence of this effect was small and highly variable between individual infants. Fifth, this study was performed in one district in Sierra Leone and so the degree of generalisability is uncertain. Additionally, the novel method limits comparisons to testing done previously in other settings, and so the significance of the results remains unclear. The study also has multiple strengths. Testing each child twice allowed for assessment of both precision in the testing and changes over time. The control infant group was appropriately age-matched and significantly different than the MAM children across all anthropometrics. The adult control group allowed for parsing the contributions of methodology and individual factors to variability. Taken together, this study demonstrates that visual orienting can be successfully assessed by eye-tracking in infants in resource-poor settings. This technique offers the potential to investigate the acute cognitive dimensions of malnutrition and its myriad effects on development robustly. The combination of its lack of relation to standard anthropometrics and its apparent responsiveness to supplemental feeding in acute malnutrition suggest it might be shedding light on a different aspect of malnutrition. ## Open access Contributors JML, KS, DTH and MJM wrote the first draft of this manuscript. JML, ASK and MJM conceptualised the study. DTH, ASK, SG and MJM selected the sites. JML, JWB and MJM developed and adapted the eye-tracking software and equipment. JML, JWB, DTH and MJM developed the trial protocol. JML, DTH, SG, BLR, PW and MJM provided supervision of the trial. JML, JWB, CG and DTH performed data collection in the field. JML, KS and MJM performed statistical analysis and interpreted the data. MJM is guarantor of the trial, data and manuscript. All authors read and approved the final manuscript. MJM affirms that the manuscript is an accurate account of the study being reported and that no important aspects of the study have been omitted. Funding This work was supported by USAID, grant AID-OAA-C-16-00020. JML, SG, CG, PW and MJM received research support from USAID AID-OAA-C-16-00020. DTH received support from National Institute of Diabetes and Digestive and Kidney Diseases under Award Number T32 DK077653. KS received support from the Knowlton Foundation at Washington University in St Louis. Competing interests None declared. Patient consent for publication Not applicable. Ethics approval The four foods trial was approved by Tufts University Human Studies Committee (ID 12313), the Washington University Human Studies Committee (ID 201610030) and the Sierra Leone Ethics and Scientific Review Committee. The study in which adult controls were recruited was approved by the Washington University Human Studies Committee and the Sierra Leone Ethics and Scientific Review Committee. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available in a public, open access repository. Data described in the manuscript, code book and analytic code were submitted to USAID's open repository and will be made publicly and freely available without restriction soon at https://data.usaid.gov. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ## Orcid ids [fig] Figure 1: Trial flow diagram. CSB+, corn-soy blend plus; CSWB, corn-soy-whey blend; MAM, moderate acute malnutrition; MUAC, mid-upper arm circumference; RUSF, ready-to-use supplementary food; SC+A, super cereal plus with amylase. *MUAC error at start was defined as mean of three MUAC measurements ≥12.5 cm or <11.5 cm. [/fig] [fig] Figure 2: Left: a child positioned for eye-tracking assessment in a field laboratory tent, powered by a solar-chargeable battery. Middle: data collector monitored the assessment outside the laboratory via real-time visualisation of eye-tracking. Right: examples of stimuli used as targets in a test assessing saccadic reaction time. [/fig] [fig] Figure 3: A) Raw (grey) and median-filtered X (red) and Y (blue) coordinates of the point of gaze on a single trial from one infant. Saccadic reaction times (SRTs) were extracted from the median-filtered data by detecting the first entry of the gaze into the area of the new saccade target. (B-E) Scatterplots showing the consistency of individual participant SRTs across splithalf subsets of trials on visit 1 (B, C), across the 4-week test-retest interval in the infant control group (D) and across split-half subsets of trials in adults (E). MAM, moderate acute malnutrition. [/fig] [fig] Figure 4: Mean saccadic reaction times (SRTs) by visit for individual infants (grey lines). Group mean is shown by the blue line. MAM, moderate acute malnutrition. [/fig] [table] Table 1: Baseline characteristics of study infants [/table] [table] Table 2: Descriptive statistics for eye-tracking measures by group and visit [/table] [table] Table 3: Differences in baseline SRT and change in SRT in children with MAM compared with controls in unadjusted and adjusted analyses [/table] [table] Table 4: Aggregate scores in the coventional tests of oculomotor function in children with MAM compared with controls Data are mean±SD and mean difference (95% CI). Independent samples t-test was used for unadjusted comparisons. Linear regression was used for adjusted comparisons. MAM, moderate acute malnutrition; SRT, saccade reaction time. [/table]

First Case Report of Developmental Bilateral Cataract with a Novel Mutation in the ZEB2 Gene Observed in Mowat-Wilson Syndrome Background: Mowat-Wilson syndrome (MWS) is extremely rare multisystemic autosomal dominant disorder caused by mutations in the Zinc Finger E-Box Binding Homeobox 2 (ZEB2) gene. Ocular pathologies are one of the symptoms that appear in the clinical picture of MWS individuals, but not many have been described so far. Pathologies such as optic nerve or retinal epithelium atrophy, iris or optic disc coloboma as well as congenital cataracts have been most frequently described until now. Therefore, we would like to report the first case of bilateral developmental cataract in a 9-year-old girl with MWS who underwent successful cataract surgery with intraocular lens implantation. Case Presentation: A 9-year-old girl, diagnosed with p.Gln694Ter mutation in ZEB2 gene and suspicion of MWS was referred to the Children's Outpatient Ophthalmology Clinic for ophthalmological evaluation. Her previous assessments revealed abnormalities of the optic nerve discs. The patient was diagnosed with atrophy of the optic nerves, convergent strabismus, and with-the-rule astigmatism. One year later, during the follow-up visit, the patient was presented with decreased visual acuity (VA), developmental total cataract in the right eye and a developmental partial cataract in the left eye. This resulted in decreased VA confirmed by deteriorated responses in visual evoked potential (VEP) test. The girl underwent a two-stage procedure of cataract removal, first of one eye and then of the other eye with artificial lens implants. In the 2 years following the operation, no complications were observed and, most remarkably, VA improved significantly. Conclusions: The ZEB2 gene is primarily responsible for encoding the Smad interaction protein 1 (SIP1), which is involved in the proper development of various eye components. When mutated, it results in multilevel abnormalities, also in the proper lens formation, that prevent the child from normal vision development. This typically results in the formation of congenital cataracts in children with MWS syndrome, however, our case shows that it also leads to the formation of developmental cataracts. This is presumably due to the effect of the lack of SIP1 on other genes, altering their downstream expression and is a novel insight into the importance of the SIP1 in the occurrence of ocular pathologies. To the best of our knowledge, this is the first case of bilateral developmental cataract in the context of MWS. Moreover, a novel mutation (p.Gln694Ter) in the ZEB2 gene was found corresponding to this syndrome entity. This report allows us to gain a more comprehensive insight into the genetic spectrum and the corresponding phenotypic features in MWS syndrome patients. # Introduction Mowat-Wilson syndrome (MWS) is a rare, multisystemic inherited disorder characterized by facial dysmorphia, psychomotor retardation, and other congenital malformations of multiple organs. It was first described in 1998 by Mowat et al. and since then, approximately 350 cases have been described worldwide [bib_ref] ZEB2 zinc-finger missense mutations lead to hypomorphic alleles and a mild Mowat-Wilson..., Ghoumid [/bib_ref]. The variety of clinical manifestations that distinguish this disease entity makes it difficult to make a uniform diagnosis; therefore, many cases are undiagnosed or diagnosed later in the child's life. The prevalence of MWS is estimated to be 1:50,00-100,000 live-born children and to date, there has been no reported case of affected individuals having offspring. Siblings are rarely reported in the literature, but their very existence may indicate possible germ cell lineage involvement and a mosaic pattern of inheritance between parents and offspring [bib_ref] Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: Delineation of a..., Mowat [/bib_ref] [bib_ref] Recurrence of Mowat-Wilson syndrome in siblings with the same proven mutation, Mcgaughran [/bib_ref]. MWS results from heterozygous mutations or deletions in the zinc finger E-boxbinding homeobox 2 (ZEB2) gene, encoding Smad Interacting Protein 1 (SIP1), which plays a key role in the transforming growth factor-beta (TGF-β) signaling pathway. This cascade is a major intracellular pathway in the human body with distinct effects on cell proliferation and differentiation. The expression of TGF-β family members is elevated during embryonic life, ensuring the promotion of normal organogenesis, including the eye [bib_ref] Transforming growth factor-beta signalling: Role and consequences of Smad linker region phosphorylation, Kamato [/bib_ref]. SIP1 expression is higher in cells derived from the neural crest, genital gland, musculoskeletal system, and eyeball. Hence, the localization and eventual damage of these cells directly correspond to the spectrum of malformations presented in MWS [bib_ref] Expression of the SMADIP1 gene during early human development, Espinosa-Parrilla [/bib_ref]. Ocular pathologies are one of the symptoms that appear in the clinical picture of MW individuals, but not many have been described so far. Pathologies such as optic nerve or retinal epithelium atrophy, iris or optic disc coloboma as well as congenital cataracts have been most frequently described until now. Therefore, we would like to report the first case of bilateral developmental cataract in a 9-year-old girl with MWS who underwent successful cataract surgery with intraocular lens (IOL) implantation. ## Case report A 9-year-old girl, diagnosed with p.Gln694Ter mutation in ZEB2 gene and suspicion of MWS was urgently referred to the Children's Outpatient Ophthalmology Clinic to expand the diagnosis of visual pathway conduction disorders and further evaluation. Her previous ophthalmic assessments revealed pallor of the temporal part of optic nerve discs. The patient was also diagnosed with convergent strabismus and astigmatism. On admission, the patient's mother presented her current medical records. The girl was born from her mother's first pregnancy and first delivery, the birth was by natural force in the 39th week of pregnancy, with a birth weight of 3440 g, body length 52 cm and head circumference 33 cm. The course of pregnancy and delivery complied with the norm. In the postnatal period, a patent ductus arteriosus and aortic valve stenosis were discovered. Furthermore, the occurrence and persistence of constipation led to the initiation of the patient's multispecialty diagnostics and the diagnosis of Hirschsprung's disease. Subsequently, due to small increase in head circumference and a noticeable delay in psychomotor development (the child does not establish contact with the environment, does not communicate, nor walk) as well as epileptic seizures the child underwent a number of diagnostic tests including head Magnetic Resonance Imaging (MRI), which revealed callous body dysgenesis, lateral and III ventricular dilatation. Due to the coexistence of the above clinical manifestations along with facial dysmorphic features characteristic for the Mowat-Wilson syndrome, the child was referred for consultation at the Genetic Clinic. Molecular testing of the coding fragment of the ZEB2 gene (exon fragment from amino acid position His655 to Ser840) detected a mutation of p.Gla694Ter in one allele. The above mutation involves a C nucleotide substitution at T at position c.2080, resulting in a change in the amino acid Gln to the STOP codon at position 694 of the amino acid chain, which ultimately leads to premature termination of translation. This type of mutation within the ZEB2 gene has not yet been described in relation to the diagnosis of MWS but in combination with the clinical picture the results spoke for it. In the further later years, epileptic seizures, premature puberty, hypertension, and significant mental and developmental disabilities appeared. The patient's father (35 years old) is healthy, her mother (30 years old) is treated for hypertension, the girl has no siblings, and the family pedigree is not burdened with genetically determined diseases. Molecular analysis of the ZEB2 gene performed on the parents did not reveal the presence of pathogenic variants, which means that the mutation in our patient occurred de novo, and the risk of the disease in subsequent children of her biological parents is low [fig_ref] Figure 1: Pedigree chart of our patient [/fig_ref]. patient's father (35 years old) is healthy, her mother (30 years old) is treated for hypertension, the girl has no siblings, and the family pedigree is not burdened with genetically determined diseases. Molecular analysis of the ZEB2 gene performed on the parents did not reveal the presence of pathogenic variants, which means that the mutation in our patient occurred de novo, and the risk of the disease in subsequent children of her biological parents is low [fig_ref] Figure 1: Pedigree chart of our patient [/fig_ref]. During the first visit, the patient's previous ophthalmology medical records were reviewed. The patient did not acquire the ability to speak and, therefore, the visual acuity (VA) could not be verified. Nevertheless, the patient followed objects with both of her eyes and the pupillary direct and indirect response to light was normal. The eyeballs remained in alternating convergent strabismus, Hirschberg test 15-20°, no limitation in eye movements was detected [fig_ref] Figure 2: Our patient with convergent strabismus and facial dysmorphia specific to MWS [/fig_ref]. During the first visit, the patient's previous ophthalmology medical records were reviewed. The patient did not acquire the ability to speak and, therefore, the visual acuity (VA) could not be verified. Nevertheless, the patient followed objects with both of her eyes and the pupillary direct and indirect response to light was normal. The eyeballs remained in alternating convergent strabismus, Hirschberg test 15-20 - , no limitation in eye movements was detected [fig_ref] Figure 2: Our patient with convergent strabismus and facial dysmorphia specific to MWS [/fig_ref]. The cycloplegic autorefraction examination revealed in the right eye (RE) −0.5 Dsph −6.0 Dcyl ax 178 - and in the left eye (LE) showed −1.0 Dsph −6.5 Dcyl ax 162 - . Anterior segment examination of both eyes was characterized by minor posterior synechiae, the optic media were translucent, and funduscopic examination confirmed the previous ophthalmologic findings and showed pallor of the optic nerve discs on the temporal side as well as a subtle inferior elevation of the optic disc in the RE. Ocular ultrasound (USG) showed a slight increase in echogenicity in the area of the optic disc, which was most likely consistent with optic nerve drusen. The patient was scheduled for ophthalmologic follow-up with a prescription of spectacle correction appropriate for her refractive error. Less than a year later, the patient had a second visit in our outpatient clinic. According to her mother's statement, for some time the girl showed worse interest in her surroundings. The patient's RE did not follow objects (neurologic and developmental causes related to the underlying disease cannot be ruled out), while LE was following objects. The pupil response to indirect and direct light, as well as the swinging light test were normal. Anterior segment eye examination revealed a total cortical cataract in the RE and a partial cortical cataract in the LE. Evaluation of the RE fundus was impossible due to total cataract, fundus of the LE was poorly visible with noticeable optic nerve rim pallor. The USG examination of the RE showed increased echogenicity in the projection of the optic nerve disc and increased echo of the lens, while in the LE it was normal [fig_ref] Figure 3: Ultrasound of the RE [/fig_ref]. An FVEP was performed to assess the patient's visual potential, but due to very difficult cooperation, it was only possible to obtain a record from the RE [fig_ref] Figure 4: FVEP of the RE [/fig_ref]. The cycloplegic autorefraction examination revealed in the right eye (RE) −0.5 Dsph −6.0 Dcyl ax 178° and in the left eye (LE) showed −1.0 Dsph −6.5 Dcyl ax 162°. Anterior segment examination of both eyes was characterized by minor posterior synechiae, the optic media were translucent, and funduscopic examination confirmed the previous ophthalmologic findings and showed pallor of the optic nerve discs on the temporal side as well as a subtle inferior elevation of the optic disc in the RE. Ocular ultrasound (USG) showed a slight increase in echogenicity in the area of the optic disc, which was most likely consistent with optic nerve drusen. The patient was scheduled for ophthalmologic follow-up with a prescription of spectacle correction appropriate for her refractive error. Less than a year later, the patient had a second visit in our outpatient clinic. According to her mother's statement, for some time the girl showed worse interest in her surroundings. The patient's RE did not follow objects (neurologic and developmental causes related to the underlying disease cannot be ruled out), while LE was following objects. The pupil response to indirect and direct light, as well as the swinging light test were normal. Anterior segment eye examination revealed a total cortical cataract in the RE and a partial cortical cataract in the LE. Evaluation of the RE fundus was impossible due to total cataract, fundus of the LE was poorly visible with noticeable optic nerve rim pallor. The USG examination of the RE showed increased echogenicity in the projection of the optic nerve disc and increased echo of the lens, while in the LE it was normal [fig_ref] Figure 3: Ultrasound of the RE [/fig_ref]. An FVEP was performed to assess the patient's visual potential, but due to very difficult cooperation, it was only possible to obtain a record from the RE [fig_ref] Figure 4: FVEP of the RE [/fig_ref]. Subsequently, cataract surgery was performed with implantation of a foldable IOL in the RE. In addition, fundus examination of the RE revealed optic nerve pallor with pigment regrouping at the retinal periphery. An identical procedure was then performed in the LE [fig_ref] Figure 5: The various steps of cataract removal and intraocular lens implantation in the... [/fig_ref]. Residual astigmatism was corrected with spectacles. Subsequently, cataract surgery was performed with implantation of a foldable IOL in the RE. In addition, fundus examination of the RE revealed optic nerve pallor with pigment regrouping at the retinal periphery. An identical procedure was then performed in the LE [fig_ref] Figure 5: The various steps of cataract removal and intraocular lens implantation in the... [/fig_ref]. Residual astigmatism was corrected with spectacles. During the follow-up, the patient was able to follow the objects again and the FVEP study results were within normal ranges. The patient is currently undergoing postopera tive 3-year follow-up and further ophthalmologic care. # Discussion MWS is a disease entity in which there are structural abnormalities of the eye and it various structures including the optic nerve and thus the pathway of conduction of visua impulses. It has been observed that the clinical features of MWS are interdependent on During the follow-up, the patient was able to follow the objects again and the FVEP study results were within normal ranges. The patient is currently undergoing postoperative 3-year follow-up and further ophthalmologic care. # Discussion MWS is a disease entity in which there are structural abnormalities of the eye and its various structures including the optic nerve and thus the pathway of conduction of visual impulses. It has been observed that the clinical features of MWS are interdependent on the type of genetic mutation. Patients with gene deletions are phenotypically similar [bib_ref] Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: Delineation of a..., Mowat [/bib_ref]. Some studies have shown that the larger the deletion, the greater the clinical impairment and the greater the similarity to typical MWS features. Unfortunately, no more than 5% of cases are caused by missense mutations of the ZEB2 gene and thus reveal a mild form of disability [bib_ref] Characterisation of deletions of the ZFHX1B region and genotype-phenotype analysis in Mowat-Wilson..., Zweier [/bib_ref] [bib_ref] Mowat-Wilson syndrome and mutation in the zinc finger homeo box 1B gene:..., Cerruti-Mainardi [/bib_ref] [bib_ref] Neuroimaging findings in Mowat-Wilson syndrome: A study of 54 patients, Garavelli [/bib_ref]. The morphological features characteristic of MWS patients are shown in table [fig_ref] Table 1: The most frequent abnormalities involving various systems found in MWS [/fig_ref]. Despite the abnormalities specific to individual systems, the overall impression is important, given that the patient is presenting for the first time. Common emblematic features are observed in 9 out of 10 cases and include a peculiar facial phenotype and posture that become more prominent with the increasing age of the patient. The face tends to be elongated and the nose is extended downward over the philtrum. The upper face is unique with epicanthic folds, orbital hypertelorism, and large, medially arched eyebrows. Characteristics of the midface include a wide nasal bridge and a saddle-shaped nose. Other suggestive features include a triangular, pointed chin, an M-shaped mouth, and unique raised earlobes with a central indentation as shown in [fig_ref] Figure 6: Characteristic features of facial dysmorphia in patients with MWS [/fig_ref]. Most patients affected by this condition have short stature. Gait limitations are usually due to central nervous system anomalies and intellectual disability. Some patients remain non-ambulatory throughout life. The sucking habit, that disappears in healthy infants, in children affected by this condition persists throughout life [bib_ref] Characterisation of deletions of the ZFHX1B region and genotype-phenotype analysis in Mowat-Wilson..., Zweier [/bib_ref] [bib_ref] Mowat-Wilson syndrome: Facial phenotype changing with age: Study of 19 Italian patients..., Garavelli [/bib_ref]. Although a few described cases have developed mildly limited speech abilities, the vast majority have profound communication impairment resulting from sparse language cognition and poor Most patients affected by this condition have short stature. Gait limitations are usually due to central nervous system anomalies and intellectual disability. Some patients remain non-ambulatory throughout life. The sucking habit, that disappears in healthy infants, in children affected by this condition persists throughout life [bib_ref] Characterisation of deletions of the ZFHX1B region and genotype-phenotype analysis in Mowat-Wilson..., Zweier [/bib_ref] [bib_ref] Mowat-Wilson syndrome: Facial phenotype changing with age: Study of 19 Italian patients..., Garavelli [/bib_ref]. Although a few described cases have developed mildly limited speech abilities, the vast majority have profound communication impairment resulting from sparse language cognition and poor motor control [bib_ref] The behavioral phenotype of Mowat-Wilson syndrome, Evans [/bib_ref]. Not many cases of ocular pathologies in MWS were identified. Those that were eventually diagnosed usually involved one eye and no correlation was found with other systemic impairments. According to Ivanovski et al. [bib_ref] Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for..., Ivanovski [/bib_ref] , in a large study carried out in the group of 87 subjects with MWS, about 10% of patients express eye problems. He commonly observed strabismus as a characteristic feature, followed by astigmatism, myopia, nystagmus, and ptosis. There were few patients described with Axenfeld anomaly, iris heterochromia, and retinal coloboma. Apart from those previously mentioned, Kilick et al. [bib_ref] A Diagnosis to Consider in Intellectual Disability: Mowat-Wilson Syndrome, Kilic [/bib_ref] presented the case of a patient with iris coloboma, Bourchany et al. [bib_ref] Clinical spectrum of eye malformations in four patients with Mowat-Wilson syndrome, Bourchany [/bib_ref] describes cataract, atrophy, or absence of optic nerve, while Ariss et al. [bib_ref] Ophthalmologic abnormalities in Mowat-Wilson syndrome and a mutation in ZEB2, Ariss [/bib_ref] reports microphthalmia and retinal aplasia [fig_ref] Figure 7: Prevalence of ophthalmic disorders in MWS [/fig_ref]. Although cataracts described in children with MWS were all congenital, no developmental cataracts have been reported so far [bib_ref] A Diagnosis to Consider in Intellectual Disability: Mowat-Wilson Syndrome, Kilic [/bib_ref] [bib_ref] Clinical spectrum of eye malformations in four patients with Mowat-Wilson syndrome, Bourchany [/bib_ref] [bib_ref] Ophthalmologic abnormalities in Mowat-Wilson syndrome and a mutation in ZEB2, Ariss [/bib_ref] [bib_ref] Mowat-Wilson syndrome, Mowat [/bib_ref]. Several other syndromes are known to manifest several features in common with MWS thereby leading to misdiagnosis. Clinical entities that should be noted include Angelman syndrome, Goldberg-Shprintzen syndrome, Pitt-Hopkins syndrome, Rumbenstein-Taybi syndrome, Smith-Lemli-Optitz syndrome, and CHARGE syndrome. Ocular pathologies are clearly manifested in CHARGE and Goldberg-Shprintzen syndrome. In the former, optic nerve head coloboma, strabismus, and amblyopia are common, while ptosis, hypertelorism, and myopia are described in patients with Goldberg-Shprintzen syndrome [bib_ref] CHARGE syndrome without colobomas: Ophthalmic findings, Dosunmu [/bib_ref]. Not many cases of ocular pathologies in MWS were identified. Those that were eventually diagnosed usually involved one eye and no correlation was found with other systemic impairments. According to Ivanovski et al. [bib_ref] Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for..., Ivanovski [/bib_ref] , in a large study carried out in the group of 87 subjects with MWS, about 10% of patients express eye problems. He commonly observed strabismus as a characteristic feature, followed by astigmatism, myopia, nystagmus, and ptosis. There were few patients described with Axenfeld anomaly, iris heterochromia, and retinal coloboma. Apart from those previously mentioned, Kilick et al. [bib_ref] A Diagnosis to Consider in Intellectual Disability: Mowat-Wilson Syndrome, Kilic [/bib_ref] presented the case of a patient with iris coloboma, Bourchany et al. [bib_ref] Clinical spectrum of eye malformations in four patients with Mowat-Wilson syndrome, Bourchany [/bib_ref] describes cataract, atrophy, or absence of optic nerve, while Ariss et al. [bib_ref] Ophthalmologic abnormalities in Mowat-Wilson syndrome and a mutation in ZEB2, Ariss [/bib_ref] reports microphthalmia and retinal aplasia [fig_ref] Figure 7: Prevalence of ophthalmic disorders in MWS [/fig_ref]. Although cataracts described in children with MWS were all congenital, no developmental cataracts have been reported so far [bib_ref] A Diagnosis to Consider in Intellectual Disability: Mowat-Wilson Syndrome, Kilic [/bib_ref] [bib_ref] Clinical spectrum of eye malformations in four patients with Mowat-Wilson syndrome, Bourchany [/bib_ref] [bib_ref] Ophthalmologic abnormalities in Mowat-Wilson syndrome and a mutation in ZEB2, Ariss [/bib_ref] [bib_ref] Mowat-Wilson syndrome, Mowat [/bib_ref]. Several other syndromes are known to manifest several features in common with MWS thereby leading to misdiagnosis. Clinical entities that should be noted include Angelman syndrome, Goldberg-Shprintzen syndrome, Pitt-Hopkins syndrome, Rumbenstein-Taybi syndrome, Smith-Lemli-Optitz syndrome, and CHARGE syndrome. Ocular pathologies are clearly manifested in CHARGE and Goldberg-Shprintzen syndrome. In the former, optic nerve head coloboma, strabismus, and amblyopia are common, while ptosis, hypertelorism, and myopia are described in patients with Goldberg-Shprintzen syndrome [bib_ref] CHARGE syndrome without colobomas: Ophthalmic findings, Dosunmu [/bib_ref]. SIP1 protein encoded by ZEB2 gene has direct and indirect influence on retinal cells development. Multiple studies define the development of the retina as a consequence of differentiation of multipotent progenitor cell into the specific progeny cell, while simultaneously, some cells are generated in direct restricted manner [bib_ref] Cell differentiation in the retina of the mouse, Young [/bib_ref] [bib_ref] Control of Retinal Development by Tumor Suppressor Genes, Cantrup [/bib_ref]. Cells generated by this process are constituents of different neural retinal layers. Specific retinal cells are differentiated with the cooperation of transcription factors that cooperate together and influence the direction and time of the differentiation [bib_ref] Cell fate determination in the vertebrate retina, Bassett [/bib_ref]. It is the cellular regulator protein by itself, but also interacts either by up or down regulation with other transcription factors. It directly facilitates generation of cells in inner neuronal and controls timing of its differentiation. These mechanisms might explain retinal pathologies arising from ZEB2 mutation in MWS patients. In the absence or defect of SIP1 protein, the faulty visual stimuli transmission is thought to arise due to loss of bipolar cells function, while rods and cones are not affected. SIP1 protein encoded by ZEB2 gene has direct and indirect influence on retinal cells development. Multiple studies define the development of the retina as a consequence of differentiation of multipotent progenitor cell into the specific progeny cell, while simultaneously, some cells are generated in direct restricted manner [bib_ref] Cell differentiation in the retina of the mouse, Young [/bib_ref] [bib_ref] Control of Retinal Development by Tumor Suppressor Genes, Cantrup [/bib_ref]. Cells generated by this process are constituents of different neural retinal layers. Specific retinal cells are differentiated with the cooperation of transcription factors that cooperate together and influence the direction and time of the differentiation [bib_ref] Cell fate determination in the vertebrate retina, Bassett [/bib_ref]. It is the cellular regulator protein by itself, but also interacts either by up or down regulation with other transcription factors. It directly facilitates generation of cells in inner neuronal and controls timing of its differentiation. These mechanisms might explain retinal pathologies arising from ZEB2 mutation in MWS patients. In the absence or defect of SIP1 protein, the faulty visual stimuli transmission is thought to arise due to loss of bipolar cells function, while rods and cones are not affected. Strabismus apparently occurs often in MWS patients. Inappropriate alignment of the eye has a wide range of etiology. In MWS, the presumptive hypothesis is for its epigenetic rather than a genetic origin, related to abnormal development of visual pathway or higher visual structures. Concomitant strabismus stands for around 90% of all cases and it is of the central origin with early onset. Improper neurodevelopment evidently leads to abnormal visual perception and strabismus. Such inadequate visual system evolution might be expressed in nystagmus and loss of binocular vision that persists or progress with patient age. Prevalence is due to abnormalities of routing of ganglionic cells and underdeveloped and immature connections between distinct parts of neurons in the visual and proprioceptive pathway; such deficits are described in patients with ZEB2 mutations [bib_ref] Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for..., Ivanovski [/bib_ref]. SIP1 expression was extensively implicated in the process of lens development. Early embryogenetic period is presented with SIP1 predominant localization in epithelium and lenticular fiber cells. While lens maturation goes forward, expression of this protein is changed and it is mainly found in the peripheral epithelial and cortical fibers, as well as in previously mentioned retinal layers. Notably, SIP1 protein is crucial in the endothelial to mesenchymal transition process, that results in the separation of the lens from the ectoderm fault in SIP1 is known to disturb cellular processes inside the developing lens before the placode become autonomous vesicle. It involves downregulation of expression of two types of proteins: βand γ-crystallins and thus alter cellular microsceletone and its desirable elastic and transparent properties [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref]. While SIP1 does not appear to regulate lens fiber cell differentiation, lens cell survival, or lens cell proliferation after lens vesicle closure, lack of SIP1 in the lens vesicle results also in faulty migration of fiber cell tips necessary for the proper packing of lens fiber cells and formation of lens sutures. These primary defects in lens development manifest as severe defects in the adult lens fiber cell morphology and organization of the meridional rows, leading to a loss of lens transparency. What corresponds to aforementioned SIP1 expression shift, was verified by Manthey et al. [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref] studies on mice with Sip1 knockout. There were two periods in lens morphogenesis implicated by timing of Sip1 loss. While Sip1 is lost on later stages of lens development it presumably alters downstream gene expression as presented in [fig_ref] Figure 8: Impact of abnormal SIP1 protein on the expression of certain genes and... [/fig_ref] [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref]. Strabismus apparently occurs often in MWS patients. Inappropriate alignment of the eye has a wide range of etiology. In MWS, the presumptive hypothesis is for its epigenetic rather than a genetic origin, related to abnormal development of visual pathway or higher visual structures. Concomitant strabismus stands for around 90% of all cases and it is of the central origin with early onset. Improper neurodevelopment evidently leads to abnormal visual perception and strabismus. Such inadequate visual system evolution might be expressed in nystagmus and loss of binocular vision that persists or progress with patient age. Prevalence is due to abnormalities of routing of ganglionic cells and underdeveloped and immature connections between distinct parts of neurons in the visual and proprioceptive pathway; such deficits are described in patients with ZEB2 mutations [bib_ref] Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for..., Ivanovski [/bib_ref]. SIP1 expression was extensively implicated in the process of lens development. Early embryogenetic period is presented with SIP1 predominant localization in epithelium and lenticular fiber cells. While lens maturation goes forward, expression of this protein is changed and it is mainly found in the peripheral epithelial and cortical fibers, as well as in previously mentioned retinal layers. Notably, SIP1 protein is crucial in the endothelial to mesenchymal transition process, that results in the separation of the lens from the ectoderm fault in SIP1 is known to disturb cellular processes inside the developing lens before the placode become autonomous vesicle. It involves downregulation of expression of two types of proteins: β-and γ-crystallins and thus alter cellular microsceletone and its desirable elastic and transparent properties [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref]. While SIP1 does not appear to regulate lens fiber cell differentiation, lens cell survival, or lens cell proliferation after lens vesicle closure, lack of SIP1 in the lens vesicle results also in faulty migration of fiber cell tips necessary for the proper packing of lens fiber cells and formation of lens sutures. These primary defects in lens development manifest as severe defects in the adult lens fiber cell morphology and organization of the meridional rows, leading to a loss of lens transparency. What corresponds to aforementioned SIP1 expression shift, was verified by Manthey et al. [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref] studies on mice with Sip1 knockout. There were two periods in lens morphogenesis implicated by timing of Sip1 loss. While Sip1 is lost on later stages of lens development it presumably alters downstream gene expression as presented in [fig_ref] Figure 8: Impact of abnormal SIP1 protein on the expression of certain genes and... [/fig_ref] [bib_ref] Loss of Sip1 leads to migration defects and retention of ectodermal markers..., Manthey [/bib_ref]. Regarding MWS patients that already underwent cataract surgery, expression of SIP1 has additional importance. Two already mentioned processes-epithelial to mesenchymal transition and epithelial lens cells differentiation into fiber cells of lens, if malfunctioning, are known to be fundamental in creation of posterior capsular opacifications. Both are known to be influenced by levels of SIP1, which in case of MWS is variable [bib_ref] The Zeb proteins δEF1 and Sip1 may have distinct functions in lens..., Manthey [/bib_ref] [bib_ref] Relationship between posterior capsule opacification and intraocular lens biocompatibility, Saika [/bib_ref]. That might result in frequently observed opacifications of the posterior lens capsule after primary cataract surgery, known as secondary cataract. Thus, cataract extraction alone may not be sufficient in VA restoration and additional procedures, including Neodymium-doped yttrium aluminium garnet (Nd:YAG) laser application, may be required. That also implies the great need of a regular, post-surgical follo-ups, allowing for frequent lens and capsule evaluation. Clearly, in the described case, the spectrum of eye abnormalities can appear as developmental but also as acquired conditions. The patient did not present typical, usually congenital rather than developmental triad of eye anomalies. There is no previous report demonstrating developmental cataract in MWS. The accurate understanding of molecular bases that drives cataract development in MWS rise the explanation for this circumstance. Although appropriate means and time were picked, the outcome of cataract surgery cannot be directly compared with otherwise healthy patients. In children with MWS, restoring the best VA by cataract removal can be extremely difficult due to the changes in morphology of the optic nerve and higher central neuronal structures described in MWS, which significantly disrupt the physiology of the visual pathway. # Conclusions Diversity of abnormalities, relevant to ophthalmological evaluation indicates multiple processes that mutation of ZEB2 gene interferes with. While a variety of potential eye pathologies are discovered and described in the literature, the entire spectrum is probably not yet fully identified and understood. Physicians should take exceptional precautions to regularly evaluate ocular structures and function in MWS patients. Further analysis is needed, comprising the management of specific eye conditions and corresponding outcomes in MWS. That should be of a great interest to rise course of future research, targeting possible molecular therapies of this entity and to create conceivable guideline paths. That will hopefully improve the detection rate of eye problems and allow for an immediate and precise medical approach to patients with MWS. Author Contributions: A.T.: conceptualization, data analysis, writing-original draft preparation, review and editing; M.Ś.: conceptualization, resources, writing-review and editing; E.F.: resources, writing-review and editing, supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. ## Institutional review board statement: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and conformed to the tenets of the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed Consent Statement: Written informed consent was obtained from the legal guardian for the publication of this paper. Due to the underlying disease, inability to write and lack of logical communication ability, consent was not obtained from the patient; the patient's legal guardian provided written consent on behalf of the patient and on his own behalf. Data Availability Statement: Not applicable. ## Conflicts of interest: The authors declare that they have no conflict of interest. [fig] Figure 1: Pedigree chart of our patient. [/fig] [fig] Figure 2: Our patient with convergent strabismus and facial dysmorphia specific to MWS. [/fig] [fig] Figure 3: Ultrasound of the RE: increased echogenicity of the lens (cataract) and a slight punctate increase in echogenicity in the projection of the optic disc (which may correspond to external drusen). [/fig] [fig] Figure 4: FVEP of the RE: latencies prolonged to 125% of norm. Left hemisphere amplitudes normal, and right hemisphere amplitudes are vestigial. [/fig] [fig] Figure 5: The various steps of cataract removal and intraocular lens implantation in the LE. [/fig] [fig] Figure 6: Characteristic features of facial dysmorphia in patients with MWS. [/fig] [fig] Medicina 2023 ,: 59, x FOR PEER REVIEW 8 of 12 [/fig] [fig] Figure 7: Prevalence of ophthalmic disorders in MWS. [/fig] [fig] Figure 8: Impact of abnormal SIP1 protein on the expression of certain genes and indirect structural defects of the lens. [/fig] [table] Table 1: The most frequent abnormalities involving various systems found in MWS. [/table]

Safety and immunogenicity of a hybrid-type vaccine booster in BBIBP-CorV recipients in a randomized phase 2 trial is a potential broad-spectrum recombinant COVID-19 vaccine that integrates the antigens from multiple SARS-CoV-2 strains into a single immunogen. Here, we evaluate the safety and immunogenicity of NVSI-06-08 as a heterologous booster dose in BBIBP-CorV recipients in a randomized, double-blind, controlled, phase 2 trial conducted in the United Arab Emirates (NCT05069129). Three groups of healthy adults over 18 years of age (600 participants per group) who have administered two doses of BBIBP-CorV 4-6-month, 7-9month and >9-month earlier, respectively, are randomized 1:1 to receive either a homologous booster of BBIBP-CorV or a heterologous booster of NVSI-06-08. The incidence of adverse reactions is low, and the overall safety profile is quite similar between two booster regimens. Both Neutralizing and IgG antibodies elicited by NVSI-06-08 booster are significantly higher than those by BBIBP-CorV booster against not only SARS-CoV-2 prototype strain but also multiple variants of concerns (VOCs). Especially, the neutralizing antibody GMT against Omicron variant induced by heterologous NVSI-06-08 booster reaches 367.67, which is substantially greater than that boosted by BBIBP-CorV (GMT: 45.03). In summary, NVSI-06-08 is safe and immunogenic as a booster dose following two doses of BBIBP-CorV, which is immunogenically superior to the homologous boost with another dose of BBIBP-CorV. T hrough the great efforts of researchers worldwide, remarkable achievements have been made in developing effective vaccines against coronavirus disease-19 . As of January 31, 2022, a total of 10 vaccines have been authorized by World Health Organization (WHO) for emergency use, and 4,084,470,843 individuals (52.1% of the population) in the world have been fully vaccinated 2 . These COVID-19 vaccines provide efficient protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the virus is continuously evolving, and a number of variants have emerged, some of which acquired immune escape capability [bib_ref] Severe acute respiratory syndrome type 2-causing coronavirus: variants and preventive strategies, Aydogdu [/bib_ref]. Due to the pandemic of SARS-COV-2 variants and the waning of immunity over time, the reports of breakthrough infections are growing [bib_ref] Severe acute respiratory syndrome type 2-causing coronavirus: variants and preventive strategies, Aydogdu [/bib_ref] [bib_ref] SARS-CoV-2 breakthrough infections in vaccinated individuals: measurement, causes and impact, Lipsitch [/bib_ref] [bib_ref] COVID-19 vaccine breakthrough infections, Gupta [/bib_ref]. The Technical Advisory Group on COVID-19 Vaccine Composition (TAG-CO-VAC) of WHO has recommended updating the composition of current COVID vaccines to develop multivalent or broad-protective vaccines against SARS-CoV-2 current and even future variants. Guided by structural and computational analysis of the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, we have designed a mutation-integrated trimeric RBD (mutI-tri-RBD) as the antigen of a recombinant COVID-19 vaccine named NVSI-06-08 (Sinopharm) [bib_ref] Design of a mutation-integrated trimeric RBD with broad protection against SARS-CoV-2, Liang [/bib_ref]. In mutI-tri-RBD, three heterologous RBDs, derived respectively from the prototype, Beta and Kappa SARS-CoV-2 strain, were connected end to end and co-assembled into a trimeric structure. By this way, mutI-tri-RBD serves as a hybrid antigen that integrates key mutations from multiple SARS-CoV-2 variants into a single protein. Pre-clinical studies have demonstrated that NVSI-06-08 elicited broader immune response against SARS-CoV-2 variants. The hybrid strategy has also been applied to HIV, coronaviruses and influenza vaccine developments, and it has been proved that hybrid-type vaccine not only can improve immune response but also effectively expand the breadth of immunity [bib_ref] Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHV challenges..., Barouch [/bib_ref] [bib_ref] Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice, Cohen [/bib_ref] [bib_ref] Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell..., Kanekiyo [/bib_ref]. Due to the waning of vaccine-induced immunity over time, an effective broad-reactive vaccine is needed as a booster dose to strengthen and broaden immunity against SARS-CoV-2 variants in the individuals who have completed a primary vaccination series. The inactivated vaccine BBIBP-CorV made by Sinopharm is one of the COVID-19 vaccines approved by WHO and has been used in many countries [bib_ref] Development of an inactivated vaccine candidate, BBIBP-CorV, with potent protection against SARS-CoV-2, Wang [/bib_ref]. The interim analysis of a phase 3 trial has demonstrated that the efficacy of two doses of BBIBP was 78.1% against symptomatic COVID-19 cases, and the occurrence of serious adverse events was rare. The geometric mean titer (GMT) of neutralizing antibodies was 156.0 on day 14 after twodose vaccinations [bib_ref] Effect of 2 inactivated SARS-CoV-2 vaccines on symptomatic COVID-19 infection in adults, Kaabi [/bib_ref]. However, several studies have shown that the neutralizing antibodies elicited by BBIBP-CorV wane over time, suggesting the need for booster vaccinations [bib_ref] Neutralizing activity of BBIBP-CorV vaccine-elicited sera against Beta, Delta and other SARS-CoV-2..., Yu [/bib_ref] [bib_ref] Humoral response to the BBIBP-CorV vaccine over time in healthcare workers with..., Badano [/bib_ref]. Given that large-scale populations worldwide have already administered two doses of BBIBP-CorV, in this trial, we evaluate the immunogenicity and safety of NVSI-06-08 as a heterologous booster dose, using homologous boost with BBIBP-CorV as control. As an exploratory study, the cross-reactive immunogenicity of the heterologous BBIBP-CorV/NVSI-06-08 prime-booster vaccination against SARS-CoV-2 variants of concerns (VOCs), including Omicron, was also assessed and compared with that of the homologous booster vaccination with BBIBP-CorV to illustrate the superior immunogenicity of NVSI-06-08 as a booster dose. # Results participants. From Oct. [bib_ref] Effects of a prolonged booster interval on neutralization of Omicron variant, Zhao [/bib_ref] to Nov. 8, 2021, a total of 1833 healthy adults (≥18 years old) were enrolled, in which 1781 (97.16%) were male. These participants were classified into three groups with different prime-boost intervals, i.e., 4-6 months, 7-9 months and >9 months. For each group, participants were randomly assigned to receive either a heterologous boost of NVSI-06-08 or a homologous boost of BBIBP-CorV. Among the enrolled participants, 1800 individuals (97.17% were male) completed booster vaccination, with 600 participants in each group [fig_ref] Figure 1: Randomization and analysis populations [/fig_ref]. All the 1800 participants who had received the booster dose of vaccination were included in Safety Set (SS) for safety analysis. A total of 1678 participants (97.32% were male) who had no protocol deviations from the follow-up visits were included in Per-protocol set (PPS) for baseline analysis and immunogenicity evaluation [fig_ref] Figure 1: Randomization and analysis populations [/fig_ref]. The majority of the participants were Bangladeshis, Indian or Pakistanis. The demographic characteristics were broadly similar between heterologous and homologous booster groups [fig_ref] Table 1: Demographic characteristics of the participants [/fig_ref]. The participants in the two groups had similar age, sex, race, height, and weight distributions. For the participants, baseline IgG concentrations and neutralizing antibody titers were quantified using a chemiluminescence enzyme immunoassay kit and the live-virus neutralization assay, respectively, before booster vaccination. The baseline antibody levels were statistically similar between the participants in heterologous and homologous booster groups. Before booster vaccination, the baseline IgG GMCs were similar in the participants among groups with different prime-boost intervals. However, neutralizing GMTs in the participants from >9-month group were lower than from 7-9-month group, and those from 7-9-month group were also lower than from 4-6-month group, which demonstrated waning of neutralizing immunity over time [fig_ref] Table 1: Demographic characteristics of the participants [/fig_ref]. The decay of neutralizing immunity highlighted the need for booster vaccination to improve the immune response. Safety. Before initiation of the trial (phase 2), a small-size phase 1 trial was firstly conducted to assess the safety of NVSI-06-08 in healthy adults. In phase 1 study, a total of 48 participants were enrolled, each of whom received three doses of NVSI-06-08 with an interval of one month between each dose. As of Jan 25, 2022, the collected data showed that within 30 days after full vaccinations, 20 (41.67%) participants reported at least one adverse event related to the study vaccine, and all the reported adverse events were grade 1 or 2. No adverse event of grade 3 and above was observed. Adverse events related to the test vaccine were mainly reported in 0-7 days. The most frequent solicited local adverse event related to the test vaccine was pain, and the solicited systemic adverse events were mainly myalgia, headache, and fatigue. No serious adverse event was reported, and no adverse event of special interest occurred. The detailed safety data of phase 1 trial are provided in Supplementary Note 1. In the phase 2 trial presented here, 146 (16.29%) participants receiving NVSI-06-08 boost and 115 (12.72%) receiving BBIBP-CorV boost reported at least one solicited adverse reaction within 7 days after vaccination, and most of them were of grade 1 or 2. The overall incidence of solicited adverse reactions was low in both booster vaccinations [fig_ref] Figure 2: Incidence and severity of solicited adverse reactions after booster vaccinations with NVSI-06-08... [/fig_ref] and Supplementary . The occurrence of solicited local adverse reactions was quite similar between heterologous and homologous booster groups. All the reported local reactions were of grade 1 or 2, and most of them were injection-site pain . Solicited systemic adverse reactions reported by participants in heterologous booster groups were also similar to those reported by homologous booster groups. The reported systemic reactions were mostly of grade 1 or 2, and the most frequent reactions were headache, muscle pain, fatigue, and fever. Grade 3 systemic reactions, including fever and muscle pain, were only observed in 0.45% participants of heterologous groups and 0.22% participants of homologous group, respectively. No grade 4 or above systemic reaction was found [fig_ref] Figure 2: Incidence and severity of solicited adverse reactions after booster vaccinations with NVSI-06-08... [/fig_ref] and Supplementary . The proportion of participants reporting unsolicited adverse reactions was also comparable between heterologous and homologous booster groups (5.36% vs 5.31%). The observed unsolicited reactions primarily included myalgia (0.56% vs 0.66%), fever (0.45% vs 0.55%) and cough (0.33% vs 0.88%), most of which were graded as level 1 or 2 (Supplementary . In both groups, no AESI and vaccination-related SAE was reported as of the time of this report. Overall, these data suggest that the heterologous boosting with a dose of NVSI-06-08 following two doses of BBIBP-CorV has a good safety profile, which was quite similar to homologous boosting with BBIBP-CorV. The detailed safety data of this trial (phase 2) are provided in Supplementary Note 2. Immunogenicity against prototype virus. According to the study protocol, a total of 23 participants with suspected symptoms were tested by PCR within 30 days after booster vaccination, but all the test results were negative. Immunogenicity analysis showed that both homologous and heterologous booster vaccinations significantly improved the neutralizing antibody titers against the prototype SARS-CoV-2 virus. However, the post-vaccination neutralizing antibody GMTs of heterologous booster group were dramatically higher than those of homologous booster groups. On day 15 post-vaccination, GMTs of neutralizing antibodies elicited by homologous BBIBP-CorV boost increased by 2.93-fold (95% CI, 2.54-3.37) in 4-6-month group, 10.34-fold in 7-9month group and 21.44-fold (18.56-24.77) in >9-month group, respectively, compared to the pre-booster baseline levels. Whereas, those elicited by heterologous NVSI-06-08 boost were significantly improved by 40. [bib_ref] Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell..., Kanekiyo [/bib_ref] A total of 1833 participants were enrolled, and 1800 received booster vaccinations. Participants were classified into three groups with different prime-boost intervals. The participants in each group were randomly assigned to receive a booster dose of eighter NVSI-06-08 or BBIBP-CorV. All the 1800 participants receiving booster vaccination were included in safety set (SS) for safety analysis. A total of 1678 participants who had no protocol deviations on follow-up visits were included in Per-protocol set (PPS) for immunogenicity analysis. after booster vaccination, and the neutralizing responses induced by heterologous boost were also remarkably superior to those by homologous boost (p < 0.0001 . Among three groups with different prime-boost intervals, the post-vaccination neutralizing antibody levels in the participants of 7-9-month group were comparable to those of >9-month group, both of which were significantly higher than those of 4-6-month group. Especially, the neutralizing GMTs elicited by NVSI-06-08 in 7-9-month group reached as high as 7719.35 against wild-type SARS-CoV-2 virus. The results suggest that a booster dose with a prime-boost interval over 6 months is immunogenically optimal. The immunogenic superiority of heterologous NVSI-06-08 booster to homologous BBIBP-CorV booster was also confirmed by anti-RBD IgG response. In line with the neutralizing antibody titers, both homologous and heterologous booster vaccinations significantly improved the RBD-specific IgG antibody levels. However, heterologous booster induced dramatically higher IgG GMCs than homologous booster in all groups with different primeboost intervals. Compared to pre-booster baselines, anti-RBD IgG GMCs increased by 2.56-3.58-fold at 15 days after vaccination in the groups boosted with BBIBP-CorV, whereas, much higher 49.15-62.62-fold increases were observed in the groups boosted with heterologous NVSI-06-08 [fig_ref] Figure 4: RBD-binding IgG antibody levels against prototype SARS-CoV-2 before and 15 and 30... [/fig_ref] Results were obtained from the participants who had no protocol deviations. Comparisons between NVSI-06-08 and BBIBP-CorV booster groups were carried out using Student's t test for continuous variables (after log-transformation for antibody titers or concentrations) and Chi-square test for non-ordered categorical variables. All the tests were two-sided and a p value < 0.05 was considered statistically significant. N the number of participants, SD standard deviation, BMI body mass index, NA neutralizing antibody, GMT geometric mean titer, GMC geometric mean concentration. Neutralizing antibody response against Omicron and other VOCs. NVSI-06-08 was designed as a hybrid-type vaccine with broader neutralizing profiles, the cross-reactive immunogenicity of heterologous NVSI-06-08 booster against multiple SARS-CoV-2 VOCs, including Omicron, was evaluated as an exploratory study. A total of 200 serum samples, collected on day 15 after booster vaccination, from the participants with sequential enrollment numbers in the 7-9-month group (99 participants receiving heterologous boost and 101 receiving homologous boost) were used in the cross-neutralizing activity tests. Both in homologous and heterologous booster groups, the neutralizing antibody level against SARS-CoV-2 Omicron variant was significantly reduced in comparison with that against the prototype strain, indicating the distinct immune-evasive ability of Omicron variant. Our results are consistent with other studies [bib_ref] Omicron variant showed lower neutralizing sensitivity than other SARS-CoV-2 variants to immune..., Ai [/bib_ref] [bib_ref] The significant immune escape of pseudotyped SARS-CoV-2 variant Omicron, Wang [/bib_ref] [bib_ref] Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2, Liu [/bib_ref] [bib_ref] Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization, Cele [/bib_ref]. However, the anti-Omicron neutralizing titers induced by heterologous boost were still significantly higher than those induced by homologous boost. In participants receiving the homologous boost of BBIBP-CorV, the neutralizing antibody GMT against Omicron variant was 45.03 (95% CI, 36.37-55.74). By comparison, in participants boosted by the heterologous NVSI-06-08, the anti-Omicron neutralizing GMT still maintained at a high level of 367.67 (95% CI, 295.50-457.47), which was 8.17fold higher than that induced by homologous boost and Supplementary . We also evaluated the neutralizing antibody response against other several SARS-CoV-2 VOCs, including Alpha, Beta, and Delta. All the tested VOCs were significantly less sensitive to the neutralization induced by BBIBP-CorV booster. However, the neutralizing antibody response against Alpha and Beta variants offered by NVSI-06-08 booster was comparable to that against prototype strain. For all the tested variants, heterologous booster induced substantially greater neutralizing antibody levels than homologous booster. The GMTs of neutralizing antibodies boosted by NVSI-06-08 were 11.04-fold, 14.98-fold, and 9.48fold higher than those boosted by BBIBP-CorV against Alpha, Beta, and Delta variants, respectively b Correspondingly, the fourfold rise rates of IgG antibodies on days 15 and 30 after boosting elicited by NVSI-06-08 booster, compared with those induced by BBIBP-CorV booster. Both in a and b, for NVSI-06-08 booster vaccination, n = 285 in 4-6-month group, n = 270 in 7-9-month group, and n = 286 in >9-month group. For BBIBP-CorV booster vaccination, n = 289 in 4-6-month group, n = 270 in 7-9-month group, and n = 278 in >9-month group. Data are presented as GMCs and 95% CIs. Differences in RBD-binding IgG antibody concentrations between heterologous and homologous booster groups were tested with two-sided grouped t test after log transformation. The fourfold rise rates between heterologous and homologous booster groups were compared by two-sided Fisher's exact test. A two-sided p value < 0.05 was considered significant. The overall safety profile of heterologous boost was quite similar to that of homologous boost, which was also comparable to the safety of the priming with two doses of BBIBP-CorV as reported previously [bib_ref] Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV: a randomised, double-blind,..., Xia [/bib_ref]. A booster dose did not distinctly increase the risk of more serious side effects. The antigen of NVSI-06-08 was designed without introducing any exogenous sequence, which did not bring additional safety risks. In the vaccine, aluminum adjuvant was used, whose safety has been verified for a long time. All these features contributed to the high safety profile of NVSI-06-08. NVSI-06-08 was designed as a hybrid-type COVID-19 vaccine with broad protection potential, which integrated multiple antigens into a single molecule. Our studies show that booster vaccination of NVSI-06-08 elicited a potent cross-neutralizing response against various SARS-CoV-2 VOCs. Especially, the neutralizing activity induced by NVSI-06-08 booster against the highly immune-evasive Beta variant was no less than that against the prototype strain, which demonstrated the immunological superiority of hybrid-type vaccine. Although the immunity offered by NVSI-06-08 booster was less effective against Omicron variant as compared to that against the prototype virus, the anti-Omicron neutralizing GMT still maintained at a considerable level of 367.67 (95% CI, 295.50-457.47) on day 15 after booster vaccination, which was substantially higher than that boosted by BBIBP-CorV. According to the increasing trends in immune response, a greater anti-Omicron neutralizing GMT can be obtained on day 30 post-boost. Given that an Omicron-specific vaccine is not yet available, the prime-boost vaccination with inactivated COVID-19 vaccine plus NVSI-06-08 may be an optional strategy against the pandemic of Omicron. The RBD of Omicron harbors numerous mutations, most of which were not integrated into the antigen of NVSI-06-08. We believe that updating the vaccine by including Omicron-carrying mutations into the immunogen should induce better immunogenicity against Omicron variant. Previous studies have indicated that hybrid-type immunogen could elicit superior B cell responses both in quantity and quality in comparison with the homologous immunogens. The superior immunogenicity of heterologous immunogen may be attributed to the avidity advantage to crossreactive B cells [bib_ref] Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell..., Kanekiyo [/bib_ref]. Considering that inactivated COVID-19 vaccines have been inoculated in large-scale populations worldwide, and the immunity offered by the vaccine decays over time [bib_ref] Neutralizing activity of BBIBP-CorV vaccine-elicited sera against Beta, Delta and other SARS-CoV-2..., Yu [/bib_ref] [bib_ref] Humoral response to the BBIBP-CorV vaccine over time in healthcare workers with..., Badano [/bib_ref] , it is of significance to choose a preferred vaccine as a booster dose to restore and even enhance the immunity against SARS-CoV-2. Our studies provided a quite effective booster strategy for the inactivated vaccine recipients. The high level of neutralizing antibodies induced by heterologous BBIBP-CorV/NVSI-06-08 vaccination could alleviate the waning of immunity and facilitate the formation of an immune barrier, which then may suppress virus mutations. The immunogenicity data show that both the absolute value and the fold rise of post-booster neutralizing antibody titers in the 7-9-month and >9-month groups were significantly greater than those in the 4-6-month group. Especially, although the prebooster baseline in the >9-month group has waned more, the post-booster neutralizing titers were still distinctly higher than those in the 4-6-month group. Our results indicate that the prime-boost vaccination with an interval over 6 months was immunogenically superior to the interval of 4-6 months. Cross-neutralizing antibody titers against SARS-CoV-2 prototype stain and several VOCs, including Alpha, Beta, Delta, and Omicron, elicited by heterologous NVSI-06-08 booster, compared with those elicited by homologous BBIBP-CorV booster. A subset of 200 serum samples, collected on day 15 post-boosting, from the participants with sequential enrollment numbers in 7-9-month group (99 participants receiving a booster dose of NVSI-06-08 and 101 receiving a third dose of BBIBP-CorV) were tested using live-virus neutralization assay. Data are presented as GMTs and 95% CIs. The GMT values are given on the graph. Differences between NVSI-06-08 and BBIBP-CorV booster groups were tested with two-sided grouped t test after log transformation. A two-sided p value < 0.05 was considered significant. ****p < 0.0001. The exact p values are presented in the figure. Many studies have revealed that prime-boost interval has a significant influence on the immune efficacy of COVID vaccines [bib_ref] Effects of a prolonged booster interval on neutralization of Omicron variant, Zhao [/bib_ref] [bib_ref] Extended interval BNT162b2 vaccination enhances peak antibody generation, Parry [/bib_ref]. Our results are consistent with those previously reported studies. An over-6-month interval may facilitate better maturation of memory cells, and improve binding affinity and production level of antibodies. This trial has several limitations. Firstly, among participants, the proportion of elderly persons aged ≥ 60 years was low, and the immune response of the BBIBP-CorV/NVSI-06-08 booster strategy in the older population should be further assessed in the future. Secondary, the number of male participants were much larger than female, and the data may not well reflect the effect in women. Thirdly, in this trial, PCR tests were conducted only for the subjects who showed any symptoms of COVID-19 or went to the hospital for treatment. It may be better to perform PCR tests at regular intervals for all the participants to monitor the incidence of COVID-19 infections after booster vaccination, which will be carried out in the following phase 3 trial. In summary, heterologous prime-boost vaccination with BBIBP-CorV plus NVSI-06-08 was well tolerated and immunogenic against not only SARS-CoV-2 prototype strain but also the VOCs including Omicron. It was immunogenically superior to the booster with the third dose of BBIBP-CorV. The findings also implied that hybrid-type vaccine could induce potent and broad immune activities, which may provide an effective strategy for broad-spectrum vaccine developments. # Methods Trial design and participants. We conducted a randomized, double-blind, controlled phase 2 trial in the United Arab Emirates (NCT05069129) to evaluate the immunogenicity and safety of NVSI-06-08 (Sinopharm) as a booster dose following a primary series of BBIBP-CorV (Sinopharm). From Oct 23 to Nov 8, 2021, participants were recruited. Trial participants included three groups of healthy adults aged ≥18 years who had received two doses of BBIBP-CorV 4-6 months, 7-9 months, and >9 months before, respectively. Written informed consent was obtained from all participants before the screening. Participants were enrolled after undergoing a health screening by inquiry, medical history review, and physical examination. Confirmed, suspected or asymptomatic COVID-19 cases were excluded from the trial. Individuals with a history of SARS or MERS infections and those vaccinated with any other COVID-19 vaccines were also excluded. The detailed inclusion and exclusion criteria are listed in the study protocol (Supplementary Note 3). Trial oversight. The trial protocol was reviewed and approved by Abu Dhabi Health Research and Technology Ethics Committee. The trial was conducted in accordance with Good Clinical Practice (GCP), the Declaration of Helsinki (with amendments) as well as the local legal and regulatory requirements, and trial safety was overseen by an independent safety monitoring committee. China National Biotec Group Co., Ltd. (CNBG) of Sinopharm was the regulatory sponsor of the trial. The trial was funded by Lanzhou Institute of Biological Products Co., Ltd (LIBP) of Sinopharm and Beijing Institute of Biological Products Co., Ltd (BIBP) of Sinopharm. National Vaccine and Serum Institute (NVSI) of Sinopharm and China National Biotec Group Co., Ltd. (CNBG) of Sinopharm designed the trial, performed the analyses, and interpreted the data. All authors had full access to study data and the corresponding authors were responsible for the decision to submit the manuscript. Studied vaccines. NVSI-06-08 is a potential broad-spectrum recombinant COVID-19 vaccine, using a hybrid mutI-tri-RBD as the antigen 7 , which was developed by the National Vaccine and Serum Institute (NVSI) of Sinopharm and manufactured by the Beijing Institute of Biological Products Co., Ltd (BIBP) of Sinopharm and Lanzhou Institute of Biological Products Co., Ltd (LIBP) of Sinopharm in accordance with good manufacturing practice (GMP). One dose of NVSI-06-08 contains 20 μg antigen and 0.3 mg aluminum hydroxide adjuvant. BBIBP-CorV is an inactivated vaccine produced by the Beijing Institute of Biological Products Co., Ltd (BIBP) of Sinopharm, which has been approved by WHO for emergency use and applied in many countries worldwide [bib_ref] Development of an inactivated vaccine candidate, BBIBP-CorV, with potent protection against SARS-CoV-2, Wang [/bib_ref]. Randomization and blinding. For allocation of the participants, a randomization list was created by the stratified blocked randomization method using the SAS software (version 9.4). Eligible participants were stratified according to the primeboost intervals, i.e., 4-6 months, 7-9 months, and >9 months. In each stratum, participants were randomly assigned in a ratio of 1:1 to either heterologous or homologous booster groups using a block randomization method with a block size of 10. A vaccine randomization list was also generated using SAS software with a randomization block size of 10. Then, participant and vaccine randomization lists were inputted into the interactive web response system (IWRS), and participants were vaccinated according to the randomization number and vaccine number obtained from IWRS. The randomization and blinding were conducted by independent personnel who were not involved in the study. Participants, investigators, and other staff remained blinded to individual treatment assignments during the trial. Procedures. At the trial site, each eligible participant received a booster vaccination of NVSI-06-08 or BBIBP-CorV through intramuscular injection in the deltoid muscle of the upper arm. After booster vaccination, participants were observed at the study site for 30 mins to identify immediate adverse reactions. Solicited adverse events (AEs) were recorded for 7 days and unsolicited AEs for 30 days postvaccination. Serious adverse events (SAEs) and adverse events of special interest (AESIs) were collected up to 6 months after a full course of immunization. The grades of local and systemic adverse events were determined according to the relevant guidance of the China National Medical Products Administration (NMPA). Nasopharyngeal swabs were collected from all the participants for PCR tests prior to booster vaccination. After the vaccination, only for the subjects who showed any symptoms of COVID-19 or went to the hospital for treatment, nasopharyngeal swabs were collected and PCR tests were conducted. Blood samples were collected before booster vaccination, and on 15 days, 30 days, 3 months, 6 months, 9 months, and 12 months after booster vaccination. Vaccination and blood collection were conducted at SKMC Center for Diabetes & Endocrinology, Abu Dhabi, UAE. The data were collected in accordance with local regulations and ICH-GCP relevant standards. Study outcomes. The primary immunogenicity outcome was the neutralizing response on 15 days and 30 days after booster vaccination, by evaluation of the geometric mean titers (GMTs) of neutralizing antibodies and the corresponding fourfold rise rate (i.e., post-/pre-boost ≥4) against SARS-CoV-2 prototype strain. Neutralizing antibody titers were measured using live-virus neutralization assay. The secondary immunogenicity outcome was geometric mean concentrations (GMCs) of IgG antibodies and the corresponding four-fold rise rate against SARS-CoV-2 prototype strain. IgG antibodies were measured using a magnetic particlebased chemiluminescence enzyme immunoassay kit. The safety outcome was the occurrence and severity of any adverse reactions within 30 days post-boost. As an exploratory study, the immunogenicity of booster vaccination against SARS-CoV-2 variants of concerns (VOCs), including Omicron, was also evaluated by the GMTs of neutralizing antibodies in a subset of participants from 7-9-month group. Neutralizing antibody titers against the VOCs, including Alpha, Beta, Delta, and Omicron, were measured using live-virus neutralization assay. Laboratory analyses. Spike receptor-binding domain (RBD)-specific IgG antibodies against the prototype SARS-CoV-2 strain were measured using a commercially available magnetic particle-based chemiluminescence enzyme immunoassay kit purchased from Bioscience (Chongqing) Biotechnology Co. (approved by the China National Medical Products Administration; approval numbers 20203400183). The IgG antibody detections were carried out on an automated chemiluminescence detector (Axceed 260) according to the manufacturer's instructions. The reference calibrator used in the kit can be traced back to WHO International Standard First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136. Neutralizing antibody titers against prototype strain and VOCs, including Alpha, Beta, Delta, and Omicron, were evaluated using live-virus neutralization assay. Serum samples were heat-inactivated at 56°C for 30 mins, and then serially diluted by twofold starting from 1:4 (in the detection of neutralizing antibodies against prototype SARS-CoV-2 strain) or 1:10 (in the detection of neutralizing antibodies against VOCs) dilution. The diluted serum was mixed with an equal volume of 100 TCID 50 of SARS-CoV-2 live virus and incubated at 37°C for 2 h. After that, the Vero cell suspension with a density of 1.5-2 × 10 5 cells per mL was added to the serum-virus mixture, and then the plates were incubated at 37°C for 3-5 days. Both cell-only and virus-only wells were also set as controls. Neutralizing antibody titer was determined as the reciprocal of the serum dilution for 50% protection against viral infection to the cell. The titer for the serum below the limit of detection was set to half value of the detection limit. The live-virus neutralization assays were performed in the BSL3 facility of the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China. SARS-CoV-2 live viruses of the prototype (QD-01), Alpha (BJ-210122-14), Beta (GD84), Delta (GD96), and Omicron (NPRC2.192100003) strains were used in the assays. All these viruses were obtained from the National Institute for Viral Disease Control and Prevention, the Chinese Center for Disease Control and Prevention (China CDC). The Vero cell used in the assay was obtained from the National Institute for Food and Drug Control (NIFDC) of China. Both RBD-binding IgG detection and live-virus neutralization assays were carried out in a blinded manner. Statistical analysis. The sample size of participants was determined by Power Analysis and Sample Size (PASS15.0) software using the expected difference between groups, predefined non-inferiority margin, intended power, significance level, and estimated dropout rate. Assuming that the fourfold rise rate after booster vaccination achieves 85%, 208 participants in each arm will be required to have 80% power to conclude non-inferiority with margin of −10% and one-sided significance level of 2.5% using Miettinen and Nurminen method. If equal GMT after booster immunization is assumed, and the standard deviation of GMT after log10 transformation is considered to be 0.7, 250 subjects in each arm will be required to have 80% power to conclude non-inferiority with a margin of 2/3 and a one-sided significance level of 2.5%. Then, considering about 15-20% drop-out rate, 600 participants are required in each of the three groups (4-6-month, 7-9-month, and >9-month groups), and 1800 subjects in total are planned to enroll. All statistical analyses were carried out using the SAS 9.4 software (SAS Institute, Cary, NC). Baseline characteristics were analyzed based on the participants who had no protocol deviations. Two-sided Student's t test and twosided Chi-square test were used, respectively, for the comparison of continuous and categorical characteristics between groups. Safety was analyzed based on the safety set (SS) that included the participants receiving booster vaccination. Safety analysis results were presented as counts and percentages of adverse reactions. The differences in safety between heterologous and homologous booster groups were analyzed using two-sided Fisher's exact test. Immunogenicity was evaluated based on the Per-protocol set (PPS), which included the participants without protocol deviation, and both the GMCs of RBD-specific IgG and GMTs of neutralizing antibodies were computed along with 95% CIs. According to the pre-booster and post-booster values, the fold rises in IgG GMCs and neutralizing antibody GMTs, as well as the associated 95% CIs, were calculated. In addition, the fourfold rise rates of IgG and neutralizing antibodies along with the associated Clopper-Pearson 95% CIs were also computed. RBD-specific IgG concentrations and neutralizing antibody titers between heterologous and homologous booster groups were compared using two-sided grouped t test after log transformation. The fold rises in IgG GMC and neutralizing GMT between two groups were also compared using two-sided grouped t test. Fourfold rise rates between heterologous and homologous booster groups were compared by two-sided Fisher's exact test. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ## Data availability The study protocol is available in the Supplementary Information file. The individual participant data will be shared after de-identification. The clinical trial is still ongoing, and the data will be available from 1 month to 1 year after the completion of the study. Researchers who provide a scientifically sound proposal will be allowed access to the individual participant data. Proposals should be directed to the corresponding author Qi Ming Li ([email protected]). The proposals will be reviewed and approved by the funder, investigator, and collaborators on the basis of scientific merit. To gain access, data requestors will need to sign a data access agreement. [fig] Figure 1: Randomization and analysis populations. [/fig] [fig] Figure 2: Incidence and severity of solicited adverse reactions after booster vaccinations with NVSI-06-08 and BBIBP-CorV, respectively. a, b Incidence and severity of local (a) and systemic (b) adverse reactions after boosted with NVSI-06-08 were compared to those boosted with BBIBP-CorV. Adverse reactions are graded according to the relevant guidance of the China National Medical Products Administration (NMPA). [/fig] [fig] Figure 3: Neutralizing antibody levels against prototype SARS-CoV-2 before and 15 and 30 days after the booster vaccinations. a GMTs of neutralizing antibodies increased from baseline (day 0) to day 15 and 30 post-boosting elicited by heterologous NVSI-06-08 booster, compared with those induced by homologous BBIBP-CorV booster. b Correspondingly, the four-fold rise rates of neutralizing antibodies on day 15 and 30 after boosting elicited by NVSI-06-08 booster, compared with those induced by BBIBP-CorV booster. Both in a and b, for NVSI-06-08 booster vaccination, n = 285 in 4-6-month group, n = 270 in 7-9-month group, and n = 286 in >9-month group. For BBIBP-CorV booster vaccination, n = 289 in 4-6-month group, n = 270 in 7-9-month group, and n = 278 in >9-month group. Data are presented as GMTs and 95% CIs. Differences in neutralizing antibody titers between heterologous and homologous booster groups were tested with two-sided grouped t-test after log transformation. The four-fold rise rates between heterologous and homologous booster groups were compared by two-sided Fisher's exact test. A two-sided p value < 0.05 was considered significant. *p < 0.05, ****p < 0.0001. The exact p values are presented in thefigure.the neutralizing GMT was improved 1.04-3.87-fold on day 30 after a booster dose of BBIBP-CorV, and more remarkably 13.19-27.31-fold after a heterologous booster dose of NVSI-06-08. Multiple lines of evidence have demonstrated that neutralizing antibody titers are highly correlated with protective efficacy against the infection of SARS-CoV-2[20][21][22] . The improvement of neutralizing antibody GMT is an indicator of enhancement of protective efficacy offered by the vaccine. Our findings indicate that a heterologous boost with NVSI-06-08 following prime vaccination with BBIBP-CorV could provide stronger protection against SARS-CoV-2 than the third dose of BBIBP-CorV.The incidence of adverse reactions was low in both heterologous and homologous booster vaccinations, and most of the reported local and systemic adverse reactions were of grade 1 or 2. [/fig] [fig] Figure 4: RBD-binding IgG antibody levels against prototype SARS-CoV-2 before and 15 and 30 days after the booster vaccinations. a GMCs of RBDbinding IgG antibodies increased from baseline (day 0) to day 15 and 30 post-boosting elicited by heterologous NVSI-06-08 booster, compared with those induced by homologous BBIBP-CorV booster. [/fig] [fig] Figure 5: Cross-neutralizing antibody titers against SARS-CoV-2 prototype stain and several VOCs, including Alpha, Beta, Delta, and Omicron, elicited by heterologous NVSI-06-08 booster, compared with those elicited by homologous BBIBP-CorV booster. A subset of 200 serum samples, collected on day 15 post-boosting, from the participants with sequential enrollment numbers in 7-9-month group (99 participants receiving a booster dose of NVSI-06-08 and 101 receiving a third dose of BBIBP-CorV) were tested using live-virus neutralization assay. Data are presented as GMTs and 95% CIs. The GMT values are given on the graph. Differences between NVSI-06-08 and BBIBP-CorV booster groups were tested with two-sided grouped t test after log transformation. A two-sided p value < 0.05 was considered significant. ****p < 0.0001. The exact p values are presented in the figure. [/fig] [table] Table 1: Demographic characteristics of the participants. [/table]

Validation of Visual Analogue Scales of job demand and job control at the workplace: a cross-sectional study Supplementary Appendix 2 Relationships between each visual analog scale and items of Karasek questionnaire Supplementary Appendix 2 Relationships between each visual analog scale and items of Karasek questionnaire 10.1136/bmjopen-2020-046403 Correlation between items from the Karasek questionnaire and each visual analog scales (job demand and job control): The result of the principal component analysis (PCA) was expressed with a representation on the first discriminant plane projection of items of Karasek scale (circle of correlations). The closer the items are to each other and far from the center, the more important is their correlation. The horizontal axis makes it possible to present patients with high values for the items (on the left) vs. patients with low values (right). The vertical axis makes it possible to determine within these patients with high values for the items, those more or less associated with the items at the top left of the quadrant of the correlation circle vs. those at the bottom left. Karasek questionnairejob control items Item 1 Item 2 Item 3 Item 4 Item 5 Item 6 Item 7 Item 8 Item 9 VAS job control 0.23** 0.21** 0.22** 0.60*** 0.28*** 0.44*** 0.32*** 0.48*** 0.27*** Karasek questionnairejob demand items Item 10 Item 11 Item 12 Item 13 Item 14 Item 15 Item 16 Item 17 Item 18 VAS job demand 0.50*** 0.58*** 0.66*** 0.45*** 0.14 0.43*** 0.44*** 0.42*** 0.25***

Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane. # Introduction Phospholipids containing polyunsaturated fatty acids are highly prone to modification by reactive oxygen species, thereby generating a plethora of biologically active oxidized phospholipids (oxPLs). 1,2 A variety of (patho-) physiological effects ascribed to oxPLs underline their relevance, e.g., in inflammation, 3-5 atherosclerosis, 6 or immune response. The modes of action are diverse. First, different families of receptors were described to become activated due to oxPL binding. 1 Second, oxPLs were shown to interact with drugs, thereby influencing their pharmacokinetics. 8 Finally, one may expect the exceptional structure of an oxPL molecule to affect its biophysical properties in the lipid membrane. In this report, we will focus on the last aspect. Bioactive oxPLs contain extensively modified or truncated acyl chains in the sn-2 position, typically terminated by polar carboxylic or aldehydic groups. As was shown by nuclear magnetic resonance, Langmuir balance, and molecular dynamics (MD) simulations, the truncated polar moiety can protrude into the aqueous phase, despite the energy penalty associated with the exposition of the nonpolar chain regions. 9-12 As a consequence, bilayer properties such as permeability, mobility, and headgroup hydration are altered by the addition of oxPLs. 13, We have recently studied the diffusional properties of a fluorescent oxPL analogue, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647), in the live cell plasma membrane, and found exceptionally high mobility of ∼2 μm 2 /s, 15 in agreement with the expected lysolipid-like behavior. Moreover, we observed transient immobilization at endocytic sites, which we attributed to a preferential partitioning within highly curved membrane regions due to the inverted cone-like shape of the PGPE-Alexa647. 15 *To whom correspondence should be addressed. Tel.: þ43-732-2468-9284. To better understand the behavior of oxPL-molecules in membranes we decided to further address its properties in welldefined model systems. We used PGPE-Alexa647 as a representative carboxylated oxPL; we have previously shown that this fluorescent analogue mimics closely the behavior of the nonlabeled molecule in living cells. All data were referenced against a conventional headgroup labeled fluorescent phospholipid, dihexadecanoylphosphoethanolamine (DHPE)-Bodipy. First, we were interested whether the probe displays any preference with respect to the phase state of the membrane. The cellular plasma membrane is believed to be segregated into domains, 17 where a more ordered ("raft-") phase shall coexist with a disordered phase. 18 Generation of phase-separated model membranes has become standard in many laboratories (for reviews see, e.g., refs 19-21) and provided a wealth of insights into the thermodynamics of lipid bilayers. 20,22 Indeed, a few reports confirmed the presence of ordered environments also in the cellular plasma membrane. In this study, we found a moderate preference of the PGPE-Alexa647 for the liquid-disordered phase but also significant partitioning into the liquid ordered phase, indicating a rather promiscuous localization. Second, in order to explain the high mobility of PGPE-Alexa647 observed in the live cell plasma membrane, we further characterized the diffusion constant in various model membranes. Experiments were performed on supported lipid bilayers (SLBs) using line-scan fluorescence correlation spectroscopy (FCS) 25 and single molecule tracking. 26 Substantially higher oxPL mobility was observed consistently; it could be diminished by increasing cholesterol content and intensified by introducing artificial obstacles. We used coarse-grained MD simulations to explain the differential mobilities. # Materials and methods Chemicals. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (dioleoylphosphatidylcholine; DOPC), sphingomyelin from porcine brain (BrSM), N-octadecanoyl-D-erythro-sphingosine (C18 ceramide; Cer), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, U.S.A.) and used without further purification. Two different fluorescent lipids were used as probes: N-(4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine and triethylammonium salt (BODIPY FL DHPE, Invitrogen, Carlsbad, CA, U.S.A.); the oxidized phopholipid 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) was synthesized as described previously. Optical Adhesive 88, used to glue the mica on coverslips, was purchased from Norland Products Inc. (Cranbury, NJ, U.S.A.). Two different buffers were used for sample preparation: buffer A (150 mM NaCl, 10 mM Hepes, and 3 mM NaN3, pH 7.4) and buffer B (PBS-buffer; PAA Pasching, Austria). Buffer A was filtered through a 0.2 μm filter (Nalgene, Rochester, NY, U.S.A.) prior to use. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (18:1 Biotinyl Cap PE; DOPE-biotin) was purchased from Avanti Polar Lipids (Alabaster, AL, U.S.A.). Supported Lipid Bilayers (SLBs). Glass slides (Menzel, #1, Braunschweig, Germany) were incubated in a 3:1 Piranha solution of sulfuric acid (J.T. Baker, 95-97%, New Jersey, U.S.A.) and hydrogen peroxide (Merck, 30%, New Jersey, USA) for 20 min, rinsed with deionized water and ethanol, and dried by nitrogen. Glass slides were then glued on a measurement chamber (Lab-Tek, Nunc, Thermo Fisher Scientific, Rochester) from which the glass support has been removed. A total of 10 mg of DOPC was dissolved in a mixture of methanol and chloroform (1:3). Then, 10 μL of a 10 mg/mL DOPC solution was evaporated under nitrogen stream and diluted with 100 μL buffer B. Vesicle solutions were prepared by ultrasonicating for 20 min. The accrued vesicle solution was put on a glass slide or avidin-coated surface. After 20 min the bilayer had been formed and was washed with buffer B. Fluorescently labeled lipids were incorporated after bilayer formation by incubation from the aqueous subphase: for this, bilayers were incubated with a 2,5 nM solution of DHPE-Bodipy and with a 5nM solution of PGPE-Alexa647 for another 20 min, followed by thorough washing with buffer B. Avidin-coated glass surfaces were prepared by incubating a Piranha-cleaned glass-coverslip with a 10 mg/mL avidin solution for 20 min, and subsequent washing with buffer B. Phase Separated Lipid Bilayers (SLBs). DOPC, bSM, and cholesterol were mixed in organic solution (1:3/methanol: chloroform) in a molar ratio of 2:2:1. After solvent evaporation, the lipid film thus obtained was slowly rehydrated using buffer B at 10 mg/mL lipid concentration and resuspended through vigorous vortexing. After sonicating the suspension at 60°C, a small aliquot was diluted in buffer A and deposited in the presence of 2 mM CaCl 2 on a ∼10 μm thick, freshly cleaved mica glued onto a glass coverslip. The coverslip was sealed with a home-built polypropylene chamber and incubated at 55°C for 5 min. After that, the sample was rinsed at the same temperature at least 10 times with buffer A and allowed to cool down to 25°C. At this stage, fluorescently labeled lipids could be incorporated into the bilayer by addition from the aqueous subphase. The concentration of DHPE-Bodipy was varied between 0.1 to 0.01 mol % and for PGPE-Alexa647 from 0.5 to 0.01 mol %. After 20 min, the sample was rinsed again to remove the fluorescent lipids from the buffer solution. Single Dye Tracing. Single molecule experiments were performed as described previously. Briefly, a Zeiss Axiovert 200 microscope was equipped with a 100Â NA=1.46 R Plan -APOCHROMAT objective (Zeiss, Oberkochen Germany). Samples were illuminated in objective-type total internal reflection (TIR) configuration via the epiport using 488 nm light from an Ar þ laser (model 2017-05AR, Spectra Physics, Mountain View, CA, U.S. A.) with an intensity of typically 9-11 kW/cm 2 and 647 nm light from a Kr þ laser (Stabilite 2017-KR, Spectra Physics) with intensities of typically 5-10 kW/cm 2 . A slit aperture (Zeiss) with a width of ∼7 μm in the object plane was used as field stop to confine the illumination area. After appropriate filtering (Z488 647MV2, Z488 647RPC, Chroma, VT, U.S.A.), emitted signals were split into two color channels using a custom-made dichroic wedge (Chroma) and imaged on the same back-illuminated, liquid-nitrogen-cooled CCD camera (Micro Max 1300-PB, ## Article Plochberger et al. Roper Scientific, Trenton, NJ, U.S.A.). For the precise control of all laser pulse trains, an acoustic-optical modulator (1205C, Isomet, Springfield, VA, U.S.A.) was used. Timing protocols were generated and controlled by an in-house program package implemented in LABVIEW (National Instruments, Austin, TX, U.S.A.). Illumination times were adjusted to values between 1 and 3 ms. Movies were recorded with a delay of 2 ms between two consecutive images. Experiments were performed in a home-built incubator box equipped with a heating unit, a temperatureadjustable stage insert and an objective heater (Pecon, Erbach, Germany). Signals were analyzed by fitting a two-dimensional Gaussian profile, yielding the position with an accuracy of ∼50 nm. The sample was mounted on a high-precision XY-stage (Scan IM 120 Â 100, M€ arzh€ auser, Germany). Data Analysis. For single molecule analysis, images were analyzed using in-house algorithms implemented in MATLAB (MathWorks, Natick, MA, U.S.A.). 27 Individual diffraction limited signals were selected and fitted with a Gaussian profile, yielding the single molecule position, the brightness B and the full width at half-maximum (fwhm) of the Gaussian function. Single molecule mobility was analyzed as described previously. In brief, trajectories are specified by a sequence of positions x B (t), with i ranging from 1 to the number of observations of this trajectory. The mean square displacements AEr 2 ae were calculated as a function of the time-lag t lag = n(t ill þ t delay ) according to AEr 2 ae = AEx B (t þ t lag )x B (t)) 2 ae, with n denoting the difference in frame index. Estimated data were analyzed by fitting with the function Molecular Dynamics Simulations. The membranes simulated in this work consisted of 2500 lipids per leaflet and contained 50 water molecules per lipid (corresponding to 12.5 water beads in the coarse grained representation). The main lipid in the bilayer is DOPC. The content of cholesterol was increased from 0% to 50% by substituting DOPC with cholesterol. Additionally, 1% of the lipid molecules was the oxidized lipid 1-palmitoyl-2-glutaroyl-snglycero-3-phosphocholine (PGPC) introduced by substituting DOPC with PGPC [fig_ref] Figure 4: Molecular dynamics simulations of DOPC bilayers containing 1% PGPC and varying concentrations... [/fig_ref]. Counterions were added randomly to electroneutralize the system. During bilayer construction the lipids were randomly placed in the plain of the bilayer and rotated using an axis normal to the membrane. Atom overlaps were resolved by first expanding the bilayer in the membrane plane, followed by a size reduction in small steps to the typical area per lipid of a DOPC-cholesterol mixture, involving energy minimization and geometry optimization for each deflation step. [bib_ref] Setting up and running molecular dynamics simulations of membrane proteins, Kandt [/bib_ref] The systems was further equilibrated by first carrying out 10 and 50 ns equilibration runs with first restraining in Z the phosphate of the phospholipids and the polar oxygen of the cholesterol and then only restraining the phospholipids. After 100 ns of unconstrained equilibration we carried out a production run of 200 ns. We used the coarse grained Martini force field. [bib_ref] The MARTINI force field: coarse grained model for biomolecular simulations, Marrink [/bib_ref] [bib_ref] Coarse grained model for semiquantitative lipid simulations, Marrink [/bib_ref] The parameters of the oxidized tail of the PGPC lipid were extracted from all atom simulations of PGPC in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using Berger lipids, 31 missing parameters were taken from the force field and from Wong-Ekkabut et al. 14 and using the SPC water model. [bib_ref] Interaction models for water in relation to protein hydration, Berendsen [/bib_ref] The truncated oxidized glycero-lipid tail was coarse grained into two beads, one containing the charged carboxy terminus, one consisting of the aliphatic carbons of the chain. The 50 ns all atom trajectory was converted into coarse grained representation. Bead distances and bond and dihedral angles were measured and added to the coarse grained description of PGPC. All simulations were performed using the Gromacs 4.0.5 MD package. [bib_ref] Algorithms for highly efficient, load-balanced, and scalable molecular simulation, Hess [/bib_ref] [bib_ref] GROMACS: Fast, flexible, and free, Van Der Spoel [/bib_ref] The integration time step was set to 20 fs. Periodic boundary conditions were applied in all dimensions. A constant temperature of 310 K was maintained using the velocity rescale (v-rescale) algorithm 35 with a coupling time of 0.3 ps, coupling independently the water and the lipids plus ions to an external bath. A pressure of 1 bar was maintained in both the membrane plane and the membrane normal, using semi-isotropic Berendsen pressure coupling scheme 36 with a time constant of 3 ps. A shift function was applied to the van der Waals interactions between 0.9 and 1.2 nm, and the electrostatic interactions were shifted over the entire range from 0 to 1.2 nm. Diffusion constants were calculated by fitting the linear part of the mean square displacment curves with AEr 2 ae = 4Dt lag , after removing overall translation by fitting of the DOPC molecules. It is a known limitation of the coarse grained Martini force field that on an absolute scale the velocity of molecules are overestimated and scaling up to an order of magnitude has been suggested. [bib_ref] The MARTINI force field: coarse grained model for biomolecular simulations, Marrink [/bib_ref] We applied a scaling factor of 15 to obtain accordance on the absolute scale of the experimentally observed diffusion rates. The density profiles were calculated using standard procedures 37,38 after removing translational shifts of the membranes, which were determined from the mean position of all DOPC headgroups. The positions of the PGPC and DOPC phosphate groups along the membrane normal were extracted Plochberger et al. Article from the density plots by fitting the phosphate group density peak with a Gaussian function [fig_ref] Figure 5: Electron density profiles extracted from the MD trajectories [/fig_ref]. Distances between each phospholipid molecule (represented by its phosphate group) and the nearest cholesterol molecule (represented by its hydroxyl group) were calculated for each time point within a trajectory [fig_ref] Figure 6: Distribution of distances r between each phospholipid molecule [/fig_ref]. The resulting histograms (binwidth 0.02 nm) were averaged for all phospholipids in the simulation. [formula] AEr 2 ae ¼ 4Dt lag þ 4σ 2ð1Þ [/formula] # Results To address the phase preference of PGPE-Alexa647, we studied its partitioning in supported lipid bilayers containing bSM:DOPC:Chol in a molar ratio of 2:2:1 [fig_ref] Figure 1: Partitioning and mobility of PGPE-Alexa647 and DHPE-Bodipy in phase-separated supported lipid bilayers [/fig_ref] , which shows separation in liquid ordered (l.o.) and disordered (l.d.) phase. [bib_ref] Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy, Kahya [/bib_ref] PGPE-Alexa647 and DHPE-Bodipy were added from the aqueous phase to ensure that the probes inserted only in the upper leaflet; flip-flop is too slow to yield probe redistribution between leaflets during the experimental time frame of less than 1 h. [bib_ref] Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by..., Kiessling [/bib_ref] While the DHPE-Bodipy was largely excluded from the ordered phase (green color channel), we observed only weak contrast for PGPE-Alexa647. We performed line-scan FCS to determine accurate values of the surface densities in the ordered (σ lo ) versus the disordered phase (σ ld ), yielding the partition coefficients K = σ lo /σ ld for PGPE-Alexa647 (K = 0.75) and for DHPE-Bodipy (K = 0.32) (see [fig_ref] Table 1: Partition Coefficients and Diffusion Constants on Phase Separated Bilayers a Standard errors... [/fig_ref]. We also calculated the diffusion constants of the probe molecules, yielding higher mobility of the oxidized phospholipid analogue versus DHPE-Bodipy in both phases [fig_ref] Table 1: Partition Coefficients and Diffusion Constants on Phase Separated Bilayers a Standard errors... [/fig_ref]. The rather high l.o.-phase partitioning of PGPE-Alexa647 lead us to the hypothesis that ordered phases in general may be well accessible by the oxPL. We thus investigated a bilayer composed of bSM:DOPC:Chol:Cer in a molar ratio of 0.64:1:1:0.36, which yields a two phase equilibrium between a ceramide-enriched gellike phase and a liquid disordered phase. [bib_ref] Effects of ceramide on liquidordered domains investigated by simultaneous AFM and FCS, Chiantia [/bib_ref] Previous data revealed efficient exclusion of all investigated fluorescent analogues. [bib_ref] Role of ceramide in membrane protein organization investigated by combined AFM and..., Chiantia [/bib_ref] We indeed confirmed reduced partitioning into the ceramide-enriched phase for both probes. Due the low partitioning, we estimated the surface densities directly from the confocal images: a partition coefficient of σ cer /σ ld = 0.004 was calculated for DHPE-Bodipy; yet, also in this case ceramide-phase partitioning of PGPE-Alexa647 was significantly higher (σ cer /σ ld = 0.01). Mobility in the l.d. phase was reduced compared to the ternary system for both probes, and the mobility difference basically vanished [fig_ref] Table 1: Partition Coefficients and Diffusion Constants on Phase Separated Bilayers a Standard errors... [/fig_ref]. Probe mobility in the ceramide-enriched gel-like phase was too low to be measurable. In summary, the oxidized lipid analogue shows rather weak phase preference; in general, its mobility is higher than the mobility of a conventional phospholipid. We continued by putting our focus on the mobility difference between PGPE-Alexa647 and DHPE-Bodipy. Glass-supported lipid bilayers of DOPC were prepared and studied at 25 or 37°C. After bilayer formation, PGPE-Alexa647 and DHPE-Bodipy were added from the aqueous phase. We used single molecule tracking to determine the diffusion constant of both lipid analogues in the same bilayer regions. To eliminate potential influences due to the immobilization of molecules at surface defects we started each recording sequence by a photobleaching pulse, which irreversibly destroyed all fluorophores in the field of view [fig_ref] Figure 2: Single molecule tracking in supported DOPC bilayers [/fig_ref]. After a recovery time of 0.5-2 s, unbleached fluorescent lipid molecules have entered the field of view and could be tracked. By this measurement mode, we could restrict the analysis to a clean fraction of freely diffusing probe molecules. For our experiments, we used stroboscopic illumination with a time delay of t del = 2 ms between two consecutive illumination pulses and an illumination time t ill = 1 or 3 ms. From the single molecule trajectories, the distribution of step sizes was determined and further analyzed to assess potential heterogeneity in the sample mobility, which would yield deviations from a monoexponential behavior. 43-45 However, we found here for all bilayers and probe molecules purely monoexponential functions (exemplified in [fig_ref] Figure 2: Single molecule tracking in supported DOPC bilayers [/fig_ref]. We also tested for anomalous subdiffusion behavior, being indicative for confinements of the tracer or nonequilibrated systems: 27 when plotting the mean square displacement (AEr 2 ae) as function of the time-lag (t lag ), anomalous subdiffusion would yield a sublinear increase. However, for all data sets we found perfectly linear (A) The upper row sketches the applied laser timing protocol, the lower row an example of an image sequence. After recording a prebleach image the according area was totally photobleached by applying a laser pulse for t bleach ∼250 ms. The photobleaching efficiency was controlled by recording an image immediately after the bleach pulse. After a recovery time of t rec ∼2000 ms, single fluorescently labeled lipids entering the field of view can be resolved as diffraction limited signals. By recording an image sequence with high time resolution (t ill = 3 ms, t delay = 2 ms) individual lipids can be tracked over multiple images for determination of their diffusion constant. Panel B shows the cumulative density function cdf of square displacements between consecutive images at t lag = 10 ms for PGPE-Alexa647. The monoexponential behavior confirms the presence of just a single mobile fraction. (C) Exact values for diffusion constants D were calculated by plotting AEr 2 ae versus t lag , and fitting with eq 1. A clear difference for D is observed by comparing PGPE-Alexa647 (dark gray line, D=7.2 ( 0.22 μm 2 /s) and DHPE-Bodipy (gray line, D=3.1 ( 0.09 μm 2 /s). Experiments were performed at 37°C. [fig_ref] Figure 2: Single molecule tracking in supported DOPC bilayers [/fig_ref]. From the slopes, the diffusion constants were calculated according to eq 1. We found substantially higher mobility for PGPE-Alexa647 compared to DHPE-Bodipy [fig_ref] Figure 3: Diffusion constant of DHPE and PGPE in a DOPC/ cholesterol bilayer as... [/fig_ref] : at 25°C the oxPL showed a ∼1.3-fold higher diffusion constant than the conventional phospholipid, at 37°C we found even a ratio of ∼2.2. Interestingly, this difference was reduced and finally disappeared for DOPC bilayers containing increasing amounts of cholesterol [fig_ref] Figure 3: Diffusion constant of DHPE and PGPE in a DOPC/ cholesterol bilayer as... [/fig_ref]. To understand this behavior, we performed coarse-grained MD simulations of a DOPC matrix containing low concentrations of PGPC and varying concentrations of cholesterol. [fig_ref] Figure 4: Molecular dynamics simulations of DOPC bilayers containing 1% PGPC and varying concentrations... [/fig_ref] shows a snapshot of the membrane: consistent with previous all-atom simulations on similar compounds, 11,13,14 we observed the reversal of the truncated acyl-chain in the sn-2 position of PGPC, which frequently protruded out of the membrane into the aqueous subphase. This phenomenon was present for all cholesterol concentrations. No aggregation or alignments of the analyzed lipids was observable on length scales >5 nm, indicating that influences of potential membrane undulations can be neglected. We determined the diffusion coefficient for both DOPC and PGPC by calculating AEr 2 ae and fitting to the linear region according to AEr 2 ae = 4Dt lag . At zero cholesterol we observed a 1.4-fold higher mobility of the oxPL PGPC compared to the matrix lipid DOPC, similar to our experimental data recorded at 25°C. Since PGPC carries essentially only one chain that is inserted into the hydrophobic core of the membrane, the consequentially small area of the lipid appears to be responsible for its high mobility. Strikingly, the simulations also revealed a convergence of the mobility ratio to 1 with increasing cholesterol concentrations [fig_ref] Figure 4: Molecular dynamics simulations of DOPC bilayers containing 1% PGPC and varying concentrations... [/fig_ref] , in perfect agreement to our experimental data. Electron density profiles gave a first hint on the origin of the effect [fig_ref] Figure 5: Electron density profiles extracted from the MD trajectories [/fig_ref]. At zero cholesterol content, the PGPC headgroup region (indicated by the phosphate group) is shifted away from the bilayer center by ∼1.5 Å compared to the DOPC headgroup region. This effect diminished with increasing cholesterol concentration down to a shift of ∼0.8 Å at 40% cholesterol. Apparently, cholesterol facilitates the incorporation of the oxPL within the lipid bilayer. The mean interaction energy between PGPC and the host matrix increased linearly with increasing cholesterol concentration [fig_ref] Figure 5: Electron density profiles extracted from the MD trajectories [/fig_ref] , indicating an improved accommodation of the oxPL in the membrane. Taken together, the simulations indicate that increasing cholesterol content leads to a shift of PGPC toward the bilayer center, which is accompanied by facilitated contacts and stronger interactions with the host lipids and consequentially an adaption of the mobility values between probe and matrix molecules. We also analyzed the average distance of DOPC or PGPC to their nearest cholesterol molecule [fig_ref] Figure 6: Distribution of distances r between each phospholipid molecule [/fig_ref]. Two peaks are clearly visible: the first one representing probe molecules with directly bound cholesterol and the second one representing probe molecules that are shielded by DOPC from the nearest cholesterol. For DOPC, two effects can be observed when increasing the cholesterol concentration: the first peak gets more pronounced, as the likelihood for direct contact with cholesterol increases, and the distance to the second peaks gets smaller, reflecting the condensing effect of cholesterol. [bib_ref] A Molecular View of the Cholesterol Condensing Effect in DOPC Lipid Bilayers, Alwarawrah [/bib_ref] [bib_ref] Areas of molecules in membranes consisting of mixtures, Edholm [/bib_ref] The plots for PGPC look qualitatively similar but reveal distinct features. First, in contrast to DOPC, the first peak is substantially lower than the second peak at low cholesterol concentrations; the ratios approach the DOPCcase at high cholesterol content. This means that PGPC has a smaller preference for being associated with cholesterol than with DOPC, most visible at low cholesterol content. Second, the location of the second peak does not shift as strongly as for DOPC, indicating that the condensing effect in the immediate proximity of PGPC is less pronounced. In summary, high cholesterol concentrations level out the differences in the mobility of oxPL and conventional lipids. It is thus difficult to reconcile the exceptionally high mobility of PGPE-Alexa647 in the plasma membrane (D = 1.3-2.4 μm 2 /s 15 ) with the high native cholesterol concentrations of 30-40%. In comparison, standard lipids or lipid-anchored proteins show a mobility which is 5-to 10-fold lower. [bib_ref] Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints, Tank [/bib_ref] [bib_ref] Both MHC class II and its GPI-anchored form undergo hop diffusion as..., Umemura [/bib_ref] [bib_ref] Dynamics of putative raft-associated proteins at the cell surface, Kenworthy [/bib_ref] We suspected that the diffusion matrix in the plasma membrane may be highly structured, providing essentially a percolation matrix. To enforce this effect in our model system, we artificially introduced obstacles in the proximal leaflet by immobilizing a substantial fraction of lipids. Interleaflet coupling should transmit the effect to the distal leaflet. [bib_ref] Transbilayer coupling of obstructed lipid diffusion in polymer-tethered phospholipid bilayers, Deverall [/bib_ref] For this, a DOPC bilayer containing 4.2 mol % Biotin-DOPE was prepared on a glass surface coated with avidin. While PGPE-Alexa647 mobility was reduced only approximately 2-fold, DHPE-Bodipy became basically immobile . Consistently, addition of 40 mol % cholesterol substantially decreased the mobility of PGPE-Alexa647 . The presence of DOPE-Biotin did not influence the mobility of PGPE-Alexa647 or DHPE-Bodipy in a DOPC bilayer on pure glass . # Discussion We studied partitioning and mobility of the oxidized phospholipid PGPE-Alexa647 with reference to the conventional phospholipid DHPE-Bodipy in different lipid membranes. In summary, we made the following observations: (i) PGPE-Alexa647 shows only marginal preference for fluid versus ordered phases. In general, conventional phospholipids show altered phase partitioning compared to one chain lipids (compare refs 54 and 55). It is tempting to follow an argument of the Vaz group, who ascribed the rate-limiting step for association of a lyso-lipid with a bilayer to the formation of free area with appropriate size in the membrane surface. [bib_ref] Kinetics and Thermodynamics of Association of a Fluorescent Lysophospholipid Derivative with Lipid..., Sampaio [/bib_ref] For the oxPL, single chain insertion is sufficient for thermodynamic equilibrium, 11 therefore the formation of a marginal free area will be sufficient for insertion of PGPE-Alexa647. The data thus indicate that both l.o. and l.d. phase show similar susceptibility for the formation of free area on the length scale of a single acyl chain cross sectional area. (ii) PGPE-Alexa647 moves faster than DHPE-Bodipy in a supported DOPC bilayer. Substantial mobility differences were observed both experimentally and theoretically. MD simulations revealed that PGPC slightly sticks out of the headgroup region, thereby essentially reducing its effective area, which most likely causes the enhanced mobility. [bib_ref] Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol lipid bilayers: a free..., Almeida [/bib_ref] The experimental data show that the effect was more pronounced at elevated temperature, indicating that the shift of PGPC out of the bilayer plane is entropically favored. [fig] 16: Moumtzi, A.; Trenker, M.; Flicker, K.; Zenzmaier, E.; Saf, R.; Hermetter, A. Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells. J. Lipid Res. 2007, 48(3), 565-82. (17) Pike, L. J. Rafts Defined. J. Lipid Res. 2006, 47(7), 1597-1598. (18) Brown, D. A.; London, E. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 1998, 164(2), 103-14. (19) de Almeida, R. F. M.; Loura, L. M. S.; Prieto, M. Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging. Chem. Phys. Lipids 2009, 157(2), 61-77. (20) Veatch, S. L.; Keller, S. L. Seeing spots: complex phase behavior in simple membranes. Biochim. Biophys. Acta 2005, 1746(3), 172-85. (21) Simons, K.; Vaz, W. L. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 269-95. (22) Feigenson, G. W. Phase boundaries and biological membranes. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 63-77. (23) Swamy, M. J.; Ciani, L.; Ge, M.; Smith, A. K.; Holowka, D.; Baird, B.; Freed, J. H. Coexisting Domains in the Plasma Membranes of Live Cells Characterized by Spin-Label ESR Spectroscopy. Biophys. J. 2006, 90(12), 4452-65. (24) Gaus, K.; Gratton, E.; Kable, E. P.; Jones, A. S.; Gelissen, I.; Kritharides, L.; Jessup, W. Visualizing lipid structure and raft domains in living cells with twophoton microscopy. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(26), 15554-9. (25) Ries, J.; Chiantia, S.; Schwille, P. Accurate determination of membrane dynamics with line-scan FCS. Biophys. J. 2009, 96(5), 1999-2008. (26) Schmidt, T.; Sch€ utz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. Imaging of single molecule diffusion. Proc. Natl. Acad. Sci. U.S.A. 1996, 93(7), 2926-9. [/fig] [fig] 27: Wieser, S.; Sch€ utz, G. J. Tracking single molecules in the live cell plasma membrane-Do's and Don't's. Methods 2008, 46(2), 131-40. [/fig] [fig] Figure 1: Partitioning and mobility of PGPE-Alexa647 and DHPE-Bodipy in phase-separated supported lipid bilayers. Panels A-C show confocal fluorescence images of a DOPC/bSM/Chol 2:2:1 supported bilayer doped with trace amounts DHPE-Bodipy and PGPE-Alexa647: an overlay, the Bodipy-channel and the Alexa647-channel are shown in panels A-C, respectively. Phase separation into l.o. and l.d. domains is clearly visible due to the exclusion of DHPE-Bodipy. We performed line-scan FCS to quantify partition coefficients and mobilities (sketch in the inset to A: the blue cone shows the detection volume, the blue and green encircled areas correspond to the l.o. and l.d. phases; the orange dotted line represents the FCS line scan). (D) Representative averaged autocorrelation curves measured in different lipid phases of the bilayer at room temperature. Shown are autocorrelation curves for the l.d. phase (upper figures) and the l.o. phase (bottom figures) for the two different probes DHPE-Bodipy (left) and PGPE-Alexa647 (right). Data were fitted for two-dimensional diffusion, yielding the diffusion constants D and the surface densities σ. The obtained fit values are listed in Table1. [/fig] [fig] Figure 2: Single molecule tracking in supported DOPC bilayers. [/fig] [fig] Figure 3: Diffusion constant of DHPE and PGPE in a DOPC/ cholesterol bilayer as a function of cholesterol content, determined by single molecule tracking. DOPC bilayers containing up to 50% cholesterol were labeled with PGPE-Alexa647 (full circles) and DHPE-Bodipy (open circles). Experiments were performed at 37°C (A) or 25°C (B). Plochberger et al. Article relationships (exemplified in [/fig] [fig] Figure 4: Molecular dynamics simulations of DOPC bilayers containing 1% PGPC and varying concentrations of cholesterol. (A) Snapshot of the system at 20% cholesterol. DOPC is show in gray, PGPC is show in blue (phosphate group in light blue), and cholesterol is show in orange. (B) Effects of increasing cholesterol on the lateral diffusion of PGPC (full circles) and DOPC (open circles). The diffusion was scaled to the experimentally determined range. [/fig] [fig] Figure 5: Electron density profiles extracted from the MD trajectories. (A) Total electron density profiles of DOPC and cholesterol are shown along with the electron density of the phosphate groups of PGPC and DOPC, which are scaled for better visualization. Data are shown for 20% cholesterol. (B) We calculated the peakshift in the maxima of the phosphate groups for DOPC versus PGPC, which is shown as function of cholesterol concentration. Error bars were determined by comparing peak position of the first half of the trajectory with the second half and by differences in peak to peak distances between upper and lower leaflets. (C) The cumulative potential energy of interaction with all membrane components per PGPC molecule is shown relative to the value obtained at 0% cholesterol content. Addition of cholesterol to the membrane increases the interaction strength while no changes where observed for interaction with the aqueous phase (not shown). (46) Goetz, R.; Gompper, G.; Lipowsky, R. Mobility and elasticity of selfassembled membranes. Phys. Rev. Lett. 1999, 82(1), 221-224. [/fig] [fig] Figure 6: Distribution of distances r between each phospholipid molecule (represented by its phosphate group) and the nearest cholesterol molecule (represented by its hydroxyl group). Plots represent averages over the entire trajectory and over all phospholipids of (A) PGPC and (B) DOPC. Bin-width is 0.02 nm. Standard deviation is shown for every 10th data point. [/fig] [table] Table 1: Partition Coefficients and Diffusion Constants on Phase Separated Bilayers a Standard errors of the mean are given in parentheses. [/table]

Sensitive and quantitative detection of cardiac troponin I with upconverting nanoparticle lateral flow test with minimized interference Measurement of cardiac troponin I (cTnI) should be feasible for point-of-care testing (POCT) to diagnose acute myocardial infarction (AMI). Lateral flow immunoassays (LFIAs) have been longimplemented in POCT and clinical settings. However, sensitivity, matrix effect and quantitation in lateral flow immunoassays (LFIAs) have been major limiting factors. The performance of LFIAs can be improved with upconverting nanoparticle (UCNP) reporters. Here we report a new methodological approach to quantify cTnI using UCNP-LFIA technology with minimized plasma interference. The performance of the developed UCNP-LFIA was evaluated using clinical plasma samples (n = 262). The developed UCNP-LFIA was compared to two reference assays, the Siemens Advia Centaur assay and an in-house well-based cTnI assay. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30-10,000 ng/L. The LoB and LoD of the UCNP-LFIA were 8.4 ng/L and 30 ng/L. The method comparisons showed good correlation (Spearman's correlation 0.956 and 0.949, p < 0.0001). The developed UCNP-LFIA had LoD suitable for ruling in AMI in patients with elevated cTnI levels and was able to quantify cTnI concentrations in patient samples. The technology has potential to provide simple and rapid assay for POCT in ED setting Cardiac troponin I (cTnI) has been used clinically, for years, for diagnosis and risk stratification in patients with suspected acute myocardial infarction (AMI) 1 . Generally, elevated levels of cTns are conjoined with damage of cardiac muscles and cTnI release to the circulation has specificity for cardiac injury 2 . Measurement of circulating levels cTnI, particularly observing the rise or fall in cTnI levels, along with the evaluation of patient symptoms, history and electrocardiographic (ECG) abnormalities are current procedures in the triage of suspected AMI patients 3,4 . The typical challenge is early identification of AMI among the large heterogenous patient population, as only one in three patients, experiencing chest pain, who visit emergency department (ED) receives AMI diagnosis 5 . Therefore, timely and correct diagnosis to rule-in AMI is important in terms of cost savings and improving the clinical outcomes of AMI patients.Point-of-care testing (POCT) can facilitate quick turn-around-times (TAT) to promote efficient diagnosis of AMI 6 . Lateral flow immunoassays (LFIAs) can be used in POCT, particularly in settings where the cost of the high-tech instrumentation may be prohibitive. LFIA in POCT is well-established, mature technology known for its low cost. However, the limitations regarding sensitivity and quantification have hindered its use in many applications 7 . In our previous work, we have described how the use of upconverting nanoparticle (UCNP) reporters in LFIAs provides quantitative results and improved sensitivity[8][9][10]. The use of UCNP measurement technology can enhance the assay sensitivity in comparison to traditional down-converting fluorescent reporters since it enables elimination of the background autofluorescence from the measurement. This is due to the unique photon upconversion luminescence of UCNP reporters converting low-energy excitation (near or at infrared)OPEN www.nature.com/scientificreports/ wavelength into high-energy emission at visible wavelengths [bib_ref] Upconverting nanoparticles, Haase [/bib_ref]. The UCNP signal can be read with a miniaturized relatively low-cost reader instruments with high performance in POCT settings. Such readers with miniaturized optics for UCNP measurement have previously been described in the literature [bib_ref] Household fluorescent lateral flow strip platform for sensitive and quantitative prognosis of..., You [/bib_ref] [bib_ref] Quantitative lateral flow strip sensor using highly doped upconversion nanoparticles, He [/bib_ref] [bib_ref] Point of care upconversion nanoparticles-based lateral flow assay quantifying myoglobin in clinical..., Ji [/bib_ref] [bib_ref] A portable and universal upconversion nanoparticle-based lateral flow assay platform for point-of-care..., Gong [/bib_ref]. The performance of LFIAs may suffer from interferences originating from the sample matrix, particularly because of the typical one or two step assay procedure with limited washing. These immunoassay (IA) interferences may be caused by different interfering agents such as circulating heterophilic IgG and IgM antibodies [bib_ref] Heterophilic antibody interference in immunometric assays, Bolstad [/bib_ref] , autoantibodies targeted against the biomarker of interest [bib_ref] False-negative immunoassay results for cardiac troponin I probably due to circulating troponin..., Bohner [/bib_ref] and human anti-animal antibodies [bib_ref] When is a heterophile antibody not a heterophile antibody? When it is..., Kaplan [/bib_ref]. In general, IgG antibodies used as binders in IAs are prone to several interfering reactions caused by circulating antibodies. If a heterophilic antibody has high affinity for the Fc-region of an IA binder antibodies, an aggregate of binder antibodies with large numbers of Fc-regions close together functions as a binding target for the heterophilic antibody [bib_ref] Heterophilic antibody interference in immunometric assays, Bolstad [/bib_ref]. Furthermore, it has been shown that complement activation by the classical pathway can occur when several IgG molecules are in close proximity, e.g. on reporter nanoparticle surface [bib_ref] C1q-mediated complement activation and C3 opsonization trigger recognition of stealth poly(2-methyl-2-oxazoline)-coated silica..., Tavano [/bib_ref] or on the solid phase [bib_ref] Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal..., Börmer [/bib_ref]. As a results, complement factors bind to the Fc region of the IA binder antibody [bib_ref] Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal..., Börmer [/bib_ref] [bib_ref] Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies, Seino [/bib_ref] which causes negative interference in the IA. Non-specific binding of autoantibodies such as rheumatoid factor (RF) has been associated with falsely elevated analyte concentrations. Since IAs utilizing nanoparticle reporters aim at extremely high sensitivities, the effect of matrix interferences should be addressed to fully utilize the potential of nanoparticle IA technologies. It has been depicted that the effects of interfering agents can be eliminated with sample pre-treatment procedures such as heating or precipitation of interfering antibodies with polyethylene glycol (PEG) [bib_ref] Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal..., Börmer [/bib_ref]. In addition, pretreatment with Ethylenediamine tetra-acetic acid (EDTA) effectively inhibits the activation of the complement pathway [bib_ref] EDTA treatment of serum unmasks complement-mediated prozone inhibition in human leukocyte antigen..., Anani [/bib_ref]. However, particularly in the case of POCT, time-consuming sample pre-treatment steps are undesirable. Therefore, in LFIAs the potential solutions should be implemented in the test device itself. In this study, we report the development of a UCNP-LFIA for cTnI. UCNP reporter technology was utilized to provide the sensitive and quantitative detection of cTnI in LFIA format to overcome the typical limitations of LFIAs. The matrix interference in plasma samples was studied and reduced by incorporating the developed sample pre-treatment steps into the LF strip. The performance of the developed UCNP-LFIA was evaluated with clinical plasma samples (n = 262). # Results Elimination of matrix interference. In LFIA strips constructed without a scrub line and EDTA in the sample pad, the addition of cTnI to LiH plasma samples led to significantly lower signal recovery as compared to what was seen with 7.5% BSA-TSA buffer. These results implied that plasma contains substances which interfere with the performance of the assay. To avoid this interference various sample pretreatment options were tested, and the results are shown in [fig_ref] Figure 1: The effect of sample pre-treatment on plasma interference [/fig_ref]. What stands out in the results is the enhancement of signal levels by all the tested pretreatment steps. While untreated plasma showed a calculated recovery of 24%, the pretreatment steps produced notable improvement in recoveries reaching values around 60-89%. LFIA strips with a scrub line and EDTA treatment induced around fivefold improvement in signals compared to the untreated LiH plasma and a 136%, recovery. As a result, anti-IgM scrub line and EDTA in the sample pad were included in all LFIA strips in the following experiments. Calibration curve, Limit of detection, and linearity. A calibration curve for the UCNP-LFIA (in the final format with the anti-IgM scrub line and EDTA supplemented sample pad) is shown in [fig_ref] Figure 2: Analytical performance of the developed proof of concept cTnI LF test, showing... [/fig_ref]. The curve was linear up to 10,000 ng/L (y = 0.76x + 2.52, R 2 = 0.996). The LoB of the assay was calculated to be 8.4 ng/L and the LoD was 30 ng/L. The linearity of the UCNP-LFIA was studied with serial dilutions of three patient samples diluted by 3 to 81-fold using blank LiH plasma pool [fig_ref] Figure 2: Analytical performance of the developed proof of concept cTnI LF test, showing... [/fig_ref]. The linear regression of the observed cTnI concentration and dilution factor showed good linearity R 2 = 0.969-996 over the measured range of 37-9 365 ng/L [fig_ref] Figure 2: Analytical performance of the developed proof of concept cTnI LF test, showing... [/fig_ref]. [fig_ref] Figure 3: Method comparison between the UCNP-LFIA and the in-house well-based reference assay [/fig_ref]. Passing and Bablok regression analysis for the UCNP-LFIA and the in-house well-based assay resulted in a slope of 1.11 (95% CI from 1.05 to 1.16) and a y-intercept of − 0.08 (95% CI from 0.02 to 0.05). The Spearman correlation coefficient was 0.956 (p < 0.0001). The mean relative difference between the two methods was 39.4% with the 95% limits of agreement ranging from − 55.1 to 134% [fig_ref] Figure 3: Method comparison between the UCNP-LFIA and the in-house well-based reference assay [/fig_ref]. A close-up on the correlation between the developed LFA and the in-house well-based assay within the cTnI concentration range of less than 100 ng/L is shown in [fig_ref] Figure 1: The effect of sample pre-treatment on plasma interference [/fig_ref]. Method comparison between the UCNP-LFIA and the Siemens Advia Centaur assay (n = 191) is shown in [fig_ref] Figure 4: Method comparison between the UCNP-LFIA and Siemens Advia Centaur TnI-Ultra reference assay [/fig_ref]. Passing and Bablok regression analysis for the UCNP-LFIA and Siemens Advia Centaur yielded in a slope of 1.07 (95% CI from 1.02 to 1.13) and a y-intercept of − 0.79 (95% CI from − 0.98 to − 0.63). The Spearman correlation coefficient was 0.949 (p < 0.0001). The mean relative difference between the two methods was − 107.7% with the 95% limits of agreement ranging from − 25.1 to − 190.4% [fig_ref] Figure 4: Method comparison between the UCNP-LFIA and Siemens Advia Centaur TnI-Ultra reference assay [/fig_ref]. # Discussion In this study, we developed an LFIA utilizing UCNP reporters to enable sensitive and quantitative detection of cTnI in LiH plasma samples with minimized interference. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30-10,000 ng/L with a read-out time of 30 min. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. Measurement of cTnI levels in point-of-care (POC) setting requires rapid assays with high sensitivity to determine whether an acute myocardial infarction (AMI) has occurred. Despite the advances in POCT technologies, detecting cTnI is still particularly challenging since cTnI assays are prone to interferences originating from biological compounds such as circulating antibodies [bib_ref] Point-of-care compatibility of ultra-sensitive detection techniques for the cardiac biomarker troponin I-Challenges..., Regan [/bib_ref]. Ideally, a POCT device for cTnI would confront these challenges, be usable outside of clinical laboratories and enable desired sensitivity and quantification. Therefore, [formula] I n T c L / g n , A I F L - P N C U [/formula] In-house well-assay, ng/L cTnI www.nature.com/scientificreports/ implementation of simple, easy-to-use tests such as lateral flow tests in POCT settings could be feasible as the lateral flow platform can be adapted with minimal training and surrounding laboratory infrastructure. The presence of autoantibodies and other interfering compounds in the sample matrix are common problems in immunoassays resulting in loss in the detection sensitivity. Here we describe approaches to reduce these kinds of interferences in an LFIA. The developed UCNP-LFIA utilized 3 + 1 configuration of Mab binders for the detection of cTnI. The selected configuration reduces the cTnI autoantibody effect as described previously [bib_ref] Troponin-specific autoantibody interference in different cardiac troponin I assay configurations, Savukoski [/bib_ref] [bib_ref] Epitope specificity and igg subclass distribution of autoantibodies to cardiac troponin, Savukoski [/bib_ref]. However, cTnI IAs are still prone to interfering effects of other biological components present in blood. IA interferences have been shown to originate from e.g., the binding of IgM and complement factors to the IA binder antibodies thus preventing the signal generation [bib_ref] Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal..., Börmer [/bib_ref] [bib_ref] Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies, Seino [/bib_ref] [bib_ref] EDTA treatment of serum unmasks complement-mediated prozone inhibition in human leukocyte antigen..., Anani [/bib_ref]. Furthermore, human RF exists in its predominant forms of IgM and IgG possibly resulting in falsely elevated analyte concentrations. Therefore, we investigated the effect of removal of both immunoglobulin types IgM and IgG from the LiH plasma. A profound improvement in signal levels and recoveries in the treated samples was observed in comparison to the untreated samples. However, the IgM/RF removal pretreatment requires more than 2 h to perform, which prevents its use in a POCT solution. Therefore, we added an EDTA treated sample pad and anti-IgM antibody scrub line dispensed on the nitrocellulose membrane to the LFIA test strips. These techniques incorporated to the test strips improved the signal recoveries without a separate pretreatment step. The findings provide valuable information on improving the analytical and clinical performance of the LFIA test devices also in general. To overcome the limitations of the conventional LFIA technology in cTnI detection, we used UCNP reporters to generate a sensitive and quantitative assay in LFIA format. The UCNP reporters have been previously shown to enable these desired properties in LFIA applications [bib_ref] Lateral flow assay for simultaneous detection of cellular-and humoral immune responses, Corstjens [/bib_ref] [bib_ref] Up-converting phosphor technology-based lateral flow assay for detection of Schistosoma circulating anodic..., Corstjens [/bib_ref]. In addition to the quantitatively measurable upconversion photoluminescence signal, the UCNPs can be used for more sensitive applications. The measurement sensitivity increases since the photon upconversion originating from the UCNP reporters can be spectrally separated from the down-converting background autofluorescence [bib_ref] Upconverting nanoparticles, Haase [/bib_ref]. For the developed UNCP-LFIA, the obtained LoD was 30 ng/L. The method comparison to the two reference assays suggests that the UCNP-LFIA could be used for the quantitative determination of cTnI. Typically, low and moderate cTnI levels around 50-100 ng/L and temporal increase in cTnI level of more than 15 ng/L in the subsequent measurements are suggestive for AMI [bib_ref] Recommendations for institutions transitioning to high-sensitivity troponin testing: JACC scientific expert panel, Januzzi [/bib_ref]. The developed LFIA concept showed potential to be used for the detection and monitoring of elevated cTnI levels in suspected AMI patients. Analytically, the developed LFIA showed a wide range of linearity, good parallelism response and precision profiles. The assay procedure of the UCNP-LFIA was simple with a TAT of 45 min. POCT for cTnI in the EDs has been shown to reduce the length of stay for patients with suspected AMI [bib_ref] Point of care troponin decreases time in the emergency department for patients..., Loten [/bib_ref]. Accelerated diagnostic protocols for AMI use serial cTnI testing with subsequent measurements of cTnI concentration to determine possible elevation in circulating cTnI concentration [bib_ref] Recommendations for institutions transitioning to high-sensitivity troponin testing: JACC scientific expert panel, Januzzi [/bib_ref]. Blood samples for the measurement of cTnI should be drawn on first assessment and repeated 3-6 h later [bib_ref] Third universal definition of myocardial infarction, Thygesen [/bib_ref] [bib_ref] Fourth universal definition of myocardial infarction, Thygesen [/bib_ref]. Thus, the results should be available within one hour TAT. To consider implementation of the developed UCNP-LFIA concept to the POCT use in clinical routine, the pre-incubation step should be integrated to the LF test device to provide a user-friendly POCT assay. Furthermore, reducing TAT from 45 min to less than 30 min would improve the UCNP-LFIA feasibility for POCT [bib_ref] Cardiac troponin assays: A review of quantitative point-of-care devices and their efficacy..., Amundson [/bib_ref]. The ability to test whole blood samples would further facilitate the POCT use of the LFIA test device. In contrast to the Siemens Advia Centaur reference assay, the UCNP-LFIA systematically gave lower cTnI values. This bias was also previously observed between the in-house well-based assay and the Siemens Advia Centaur TnI-Ultra reference assay 32 . Antibodies targeting different epitopes in different assays may result in differences in the measured cTnI values [bib_ref] Troponin-specific autoantibody interference in different cardiac troponin I assay configurations, Savukoski [/bib_ref]. In addition, differences may arise from differences in calibration as standardization of cTnI assays has not been successful [bib_ref] Counterpoint: Standardization of cardiac troponin I assays will not occur in my..., Apple [/bib_ref] [bib_ref] Toward standardization of cardiac troponin I measurements part II: Assessing commutability of..., Christenson [/bib_ref]. The similarity between the UCNP-LFIA and the inhouse well-based assay can be explained by the fact that these assays share a very similar 3 + 1 antibody design and utilize the same calibration material [bib_ref] Chimeric recombinant antibody fragments in cardiac troponin I immunoassay, Hyytiä [/bib_ref]. The results of our evaluation show that the developed UCNP-LFIA technology can be used to detect cTnI in plasma in concentrations relevant in AMI diagnostics. The clinical utility of the developed test should be further evaluated with a cohort of healthy controls and with samples from patients with chest pain visiting EDs. Furthermore, LFIA strip manufacturing and reporter drying process is known to be critical for the test performance even in industrial scale. Therefore, the final clinical performance of the test, including the determination proper reference ranges of the UCNP-LFIA, should be evaluated with a finally optimized production prototype of the UCNP-LFIA manufactured with controlled and reproducible processes. To sum up, detection of cTnI for diagnosis of AMI requires both high sensitivity and accurate quantitation. In this study, we developed a proof-of-concept UCNP-LFIA for sensitive detection of cTnI and demonstrated with patient samples that the UCNP-LFIA technology can be used for this type of demanding quantitative analyses. Such LFIA could be used for aiding in diagnosis of AMI patients at the ED departments in POC settings, particularly in resource-limited settings where central laboratory diagnostics or high-cost POCT instrumentation is not feasible. The sample pretreatment options applied in this study can provide valuable information for LFIA development also for other analytes requiring high analytical and clinical performance. . The antibody binding on cTnI epitopes is illustrated in [fig_ref] Figure 5: A schematic illustration of the cTnI epitopes and the binding sites of... [/fig_ref]. A goat polyclonal anti-IgM (μ-chain specific) antibody (product# I1636) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Human cardiac troponin complex (I-T-C) (#8T62) was obtained from HyTest Ltd, and the cTnI calibrators were prepared by diluting this complex to blank LiH plasma pool. The blank LiH plasma pool and serum pool from the same individuals was compared to cTnI free serum (HyTest) to confirm signal levels equal to cTnI free serum in both pools. # Materials and methods ## Antibody bioconjugation of upconverting nanoparticles. carboxylated upcon™ ucnp-reporter particles of 78 nm diameter with a hydrophilic coating (Kaivogen Oy, Turku, Finland) were covalently coupled with the Mab 625. Briefly, the UCNP solution containing 0.5 mg UCNPs was centrifuged for 30 min at 20,000×g at room temperature (RT). The supernatant was removed, and the particle surface was activated by suspending the pellet into 135 µL of 50 mM MES containing 2 mM EDC (Sigma-Aldrich) and 30 mM sulfo-NHS (Sigma-Aldrich). The incubation was performed in shaking for 15 min at RT. The UCNPs were washed by centrifugation for 10 min at 20,000×g at RT, removing the supernatant and suspending the UCNPs to 20 mM MES, pH 6.1. The UCNPs were centrifuged as before and resuspended into 50 mM MES, pH 6.1 containing 30 µg of Mab 625 and 100 mM NaCl with the total volume of 500 µL. This conjugation reaction was incubated for 30 min at room temperature in shaking and the reaction was stopped by adding glycine, pH 11, to a final concentration of 50 mM. cTnI-UCNP lateral flow assay. The UCNP-LFIA procedure was initiated by mixing 25 µL of sample and 25 µL of reporter solution (20 ng UCNP diluted in wash buffer solution containing 0.05 M Tris pH 7.5, 0.5 M NaCl, 0.04% NaN 3 , 2 mM KF, 1.5% BSA, 0.06% bovine IgG, 0.2 mg/mL mouse IgG and 0.05 mg/mL denatured mouse IgG) and pre-incubating for 15 min at + 35 °C in slow shaking. First, the total volume of 50 µL of the mixed sample and reporter solution was allowed to absorb to the sample pad of the strip followed by immediate addition of 50 µL of wash buffer. The liquid was allowed to absorb to the strip for 30 min before measurement. The strips were read with an Upcon reader device (Labrox Oy, Turku, Finland). The UCNPs were excited at 976 nm and the emission was measured at 540 nm with a Z-focus point of 8 mm over the range of 12 mm or 125 scanning points. Emission spot size was adjusted at 1 mm for the lateral flow strips. The assay procedure is illustrated in [fig_ref] Figure 6: Schematic illustration of the protocol of the developed lateral flow assay and... [/fig_ref]. Matrix interference studies. The elimination of interference was first studied with LFIA strips that did not contain the scrub line. The removal of the RF and IgM from the serum/plasma sample prior to addition into the LF strips was investigated by using the commercial clean up solution (ab215121, Abcam, Cambridge, UK). The pretreatment was done as follows: the solution was diluted to the sample in a 1:10 ratio. The samples were incubated at 4 °C for 1 h, prior to centrifugation at 700×g for 45 min at 4 °C. The supernatant was carefully transferred to a fresh tube, to avoid any formed pellets. Samples were immediately assayed according to the developed UCNP-LFIA protocol described above. The signal levels obtained from LiH plasma pool spiked with 2000 ng/L cTnI (I-T-C troponin complex) were compared to those obtained from spiked 7.5% BSA-TSA buffer and spiked untreated LiH plasma. The impact of EDTA on the performance was studied by drying 50 mM EDTA to the sample pad. To further investigate the practical means of avoiding the matrix interference, anti-IgM scrub line was added to the LFIA strips as described above. www.nature.com/scientificreports/ Assay evaluations. The limit of blank (LoB) and limit of detection (LoD) for the final assay design were determined according to the Classical Approach of CLSI Guideline EP17-A2 using above described blank LiH plasma pool as a zero calibrator and five LiH plasma pools with spiked cTnI concentrations of 1-4 × LoB. Initial LoB was determined with 20 replicates of blank pool samples. The blank pools and low concentration samples were run during 4 days in 5 replicates each. Due to the non-normal distribution of the blank sample results, a non-parametric data analysis was used. For low concentration samples, the non-parametric data analysis approach was used because of the non-normal distribution among the results from these samples. In the nonparametric analysis, LoD was determined as the median value of the three highest low concentration samples (total number of the replicates n = 60) yielding in more than 95% positive scores above LoB. The calibration curve was determined with cTnI concentrations of 5-200,000 ng/L. Each concentration was run in three replicate strips. The calibration curve was run in each testing day in parallel with patient samples to assess daily variations. The curves were fitted by using linear regression for logarithmic values. The patient samples (n = 262) were assayed with the UCNP-LFIA procedure in triplicates and the sample concentrations were calculated against the calibration curve run at the same time with the evaluated set of samples. Parallelism was assessed by serial dilution of three LiH plasma samples with endogenous cTnI concentrations of 9365, 7329 and 3060 ng/L (measured with the in-house well-based assay). Each sample was diluted up to 1/81 with blank LiH plasma pool and each dilution was measured in three replicates. Statistical analysis. Statistical analyses were performed with IBM SPSS Statistics 21 (SPSS Inc., Chicago, IL, USA). Noted p values were 2-tailed, and all p values < 0.05 were considered statistically significant. Normality of the distribution of continuous variables was determined by the Shapiro-Wilk test and visual inspection. For the clinical samples, assay descriptive and Spearman's rank correlation coefficients were calculated. Passing and Bablok regression analysis (MedCalc Software, MedCalc Software Ltd., Ostend, Belgium) was used when studying linear regression between the UCNP-LFIA and the reference method. The linearity of sample dilutions was assessed with linear regression analysis. ## Data availability The datasets generated and analyzed during the current study can be made available by the corresponding author upon reasonable request. [fig] Figure 1: The effect of sample pre-treatment on plasma interference. All samples were analysed as unspiked (blank) and spiked with 2000 ng/L of cTnI before the pre-treatment. Pooled EDTA plasma samples were used in this experiment. IgM and RF commercial clean up solution was compared to 250 mM EDTA, IgM scrub line (prior to test line) and to different combinations. The error bars represent standard deviations of three replicate LFIA strips.Method comparison. Method comparisons were conducted using LiH-plasma samples with varying cTnI concentrations (n = 262). Samples below the LoD and outside the linear range of the assays were excluded from the comparisons. The method comparison between the UCNP-LFIA and the in-house well-based assay (n = 188) is shown in [/fig] [fig] Figure 2: Analytical performance of the developed proof of concept cTnI LF test, showing the standard curve, precision profile and parallelism. (A) Calibration curve for the UCNP-LFIA. The error bars represent the standard deviation of three replicate strips. Equation of the curve was y = 0.76x + 2.52, R2 = 0.996. (B) Precision profile determined from the calibration curve measurements. CV% describes the variation between the calculated cTnI concentrations. (C) Parallelism of the UCNP-LFIA. Three clinical LiH-plasma samples containing variable amounts of endogenous cTnI were serially diluted to 1/81-fold with blank LiH-plasma pool. [/fig] [fig] Figure 3: Method comparison between the UCNP-LFIA and the in-house well-based reference assay (n = 188). (A) Correlation of the assays and (B) Bland-Altman analysis of agreement. The relative difference is calculated as UCNP-LFIA concentration subtracted by in-house well-based assay concentration divided by mean concentration. The mean difference (39.4%) is presented with a solid line and the 95% limits of agreement (from − 55.1 to 134%) are shown with dashed lines. [/fig] [fig] Figure 4: Method comparison between the UCNP-LFIA and Siemens Advia Centaur TnI-Ultra reference assay (n = 191). (A) Correlation of the assays and (B) Bland-Altman analysis of agreement. [/fig] [fig] Figure 5: A schematic illustration of the cTnI epitopes and the binding sites of the antibodies in the developed LFIA. The developed UCNP-LFIA utilized the (3 + 1) assay design, with three capture antibodies against the N-terminus, midfragment and C-terminus of cTnI and one tracer antibody against the C-terminus of cTnI molecule. Created with www. BioRe nder. com. [/fig] [fig] Figure 6: Schematic illustration of the protocol of the developed lateral flow assay and the strip design. Created with www. BioRe nder. com. Scientific Reports | (2021) 11:18698 | https://doi.org/10.1038/s41598-021-98199-y [/fig]

The Emerging Applications of Nanotechnology in Neuroimaging: A Comprehensive Review Neuroimaging modalities such as computer tomography and magnetic resonance imaging have greatly improved in their ability to achieve higher spatial resolution of neurovascular and soft tissue neuroanatomy, allowing for increased accuracy in the diagnosis of neurological conditions. However, the use of conventional contrast agents that have short tissue retention time and associated renal toxicities, or expensive radioisotope tracers that are not widely available, continue to limit the sensitivity of these imaging modalities. Nanoparticles can potentially address these shortcomings by enhancing tissue retention and improving signal intensity in the brain and neural axis. In this review, we discuss the use of different types of nanotechnology to improve the detection, diagnosis, and treatment of a wide range of neurological diseases. # Introduction Current non-invasive modalities used in neuroimaging and diagnosis of diseases of the central nervous system (CNS) include: computed tomography (CT), magnetic resonance imaging (MRI), functional MRI (fMRI), functional near-infrared spectroscopy, functional photoacoustic imaging, and molecular imaging including positron emission tomography (PET), single photon emission CT (SPECT), and molecular MRI [bib_ref] Introduction to Neuroimaging, George [/bib_ref]. While conventional CT and MRI are by far the workhorses in neuroradiology, limitations of these techniques exist, including: the relative low sensitivity and tissue resolution of CT, the long acquisition time required for MRI, and the high cost of radioisotopes and low resolution of PET [bib_ref] Introduction to Neuroimaging, George [/bib_ref]. Furthermore, common gadoliniumbased (Gd) contrast agents such as gadopentetate dimeglumine (Magnevist ® , , administered intravenously to allow for enhancement and better visualization of both intra-and extra-axial brain tumors, have a short half-life in the bloodstream, low retention time in the tumor microenvironment, and increased risk of nephrotic systemic fibrosis in patients with renal insufficiency. To overcome these limitations, researchers have began incorporating the use of nanotechnology to enhance the ability of these agents to achieve longer retention time in the CNS, better spatial resolution on imaging, and delivery of therapies to the cells of interest in the brain [bib_ref] The Application of Nanotechnology for the Diagnosis and Treatment of Brain Diseases..., Ngowi [/bib_ref]. The diverse biophysical and chemical properties of nanoparticles (NPs) are distinct advantages for their use in CNS applications. Their small size (usually 1-100 nm in largest diameter), modularity, diverse chemical compositions, and ability to be functionalized with surface moieties to allow for targeting across the blood-brain barrier (BBB) to a specific cell type of interest, makes NPs an attractive carrier for the delivery of cargo into the CNS for use in downstream biomedical applications [bib_ref] Nanotechnology and its Use in Imaging and Drug Delivery (Review), Sim [/bib_ref]. In this review, we will discuss the emerging use of nanotechnology to assist in the imaging of neurological conditions including: 1) Primary and secondary brain tumors; 2) Cerebrovascular disorders; 3) Neurodegenerative disorders; and 4) Functional neurological disorders. ## Nanotechnology for the imaging of brain tumors MRI with and without the use of Gd contrast allows for the delineation of higher grade primary brains tumors such as glioblastoma, secondary brain metastases, and extra-axial tumors such as meningiomas, due to the nature of these tumors to secrete vascular endothelial growth factor which causes the formation a tortuous and leaky BBB around the tumor which allows for the extravasation of contrast agent, leading to visualization of the tumor mass on subsequent T1weighted MRI sequences [bib_ref] Angiogenesis in Brain Tumours, Jain [/bib_ref] [bib_ref] Introduction to Neuroimaging, George [/bib_ref]. However, conventional contrast-enhanced MRI sequences are limited in being able to differentiate the phenomenon of pseudoprogression (caused by radiation changes to surrounding tumor vasculature increasing leakiness and subsequent enhancement on post-treatment MRI scans) from true tumor progression. To increase the spatial resolution of tumors on MRI, magnetic NPs such as iron oxide nanoparticles (IONPs) or manganese oxide nanoparticles (MnO NPs) have demonstrated increased relaxivity on T1-and T2-weighted MRI sequences [bib_ref] Magnetic Nanoparticles: Current Trends and Future Aspects in Diagnostics and Nanomedicine, Vallabani [/bib_ref]. Functionalization of IONPs and MnO NPs with folate or albumin have enabled specific targeting of glioma cells in orthotopic mouse models of glioma, enabling longer periods of signal enhancement on MRI and could possibly aid in differentiating between true tumor signal versus pseudoprogression [bib_ref] Folic Acid-Conjugated MnO Nanoparticles as a T1 Contrast Agent for Magnetic Resonance..., Chen [/bib_ref] [bib_ref] An Albumin-Binding T1-T2 Dual-Modal MRI Contrast Agents for Improved Sensitivity and Accuracy..., Zhou [/bib_ref]. Other researchers have synthesized Gd-chelated-IONPs functionalized with interleukin-13 [bib_ref] A New Interleukin-13 Amino-Coated Gadolinium Metallofullerene Nanoparticle for Targeted MRI Detection of..., Li [/bib_ref] , antibodies against the mutant epidermal growth factor receptor (EGFRvIII) [bib_ref] EGFRvIII Antibody-Conjugated Iron Oxide Nanoparticles for Magnetic Resonance Imaging-Guided Convection-Enhanced Delivery and..., Hadjipanayis [/bib_ref] , or cyclic arginine-glycine-aspartic acid (cRGD) peptides [bib_ref] The Role of Astrocyte Dysfunction in Parkinson's Disease Pathogenesis, Booth [/bib_ref] , to enhance glioma targeting for enhanced imaging in preclinical animal models of glioma. A recent clinical trial demonstrated the ability of the FDA-approved IONP ferumoxytol conjugated to gadolinium contrast to define glioma pseudoprogression on MRI [bib_ref] Combined Iron Oxide Nanoparticle Ferumoxytol and Gadolinium Contrast Enhanced MRI Define Glioblastoma..., Barajas [/bib_ref] , while another pilot study demonstrated the ability of feromoxytol to detect tumor-associated macrophages on enhanced MRI [bib_ref] Quantification of Macrophages in High-Grade Gliomas by Using Ferumoxytol-Enhanced MRI: A Pilot..., Iv [/bib_ref] , showing the potential for IONP enhanced MRIs to improve cellular resolution of brain tumors. Current clinical trials investigating the use of IONPs for molecular imaging of brain tumors include: 1) A phase I safety trial of intravenous ferumoxytol for dynamic MR imaging of recurrent high-grade gliomas patients on chemotherapy (NCT00769093) ; 2) A phase I study of ferumoxytol to assess early treatment response using perfusion MRI in patients with glioblastoma (NCT00660543) ; 3) A phase 0 safety and effectiveness exploratory trial of ferumoxytol in assessing vascular properties of pediatric brain tumors in a single imaging session (NCT00978562); 4) A phase 2 study comparing ferumoxytol in characterizing tumor vasculature compared to Gd-contrast MRI in 3 and 7 T MRI machines in patients with malignant brain tumors (NCT00659126); 5) A phase 2 study of ferumoxytol in characterizing tumorassociated vasculature in patients with primary brain tumors or lung or breast brain metastases using MRI (NCT00103038) ; and 6) A phase 2 study of the safety of ferumoxytol in causing CNS inflammation (NCT00659776) (OHSU, 2022). ## Nanotechnology for the diagnosis of neurodegenerative disorders Neurodegenerative disorders encompass a wide variety of neurological conditions, from Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD), to Multiple Sclerosis (MS) and motor neuron diseases. As a full discussion of all these disorders goes beyond the scope of this review, we will focus on AD and PD-the two neurodegenerative disorders for which there have been the most prolific application of NPs as a neuroimaging tool to aid in diagnosis and management. ## I) alzheimer's disease The two pathognomonic features of AD include extracellular beta-amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau protein in dystrophic neurites (Alzheimer's . Current imaging modalities for the diagnosis of Aβ plaques and tau tangles include radioactive PET tracers such as fluorodeoxyglucose, [ 18 F] flutemetamol, and [ 18 F]flortaucipir [bib_ref] Diagnostic Tests for Alzheimer's Disease: Rationale, Methodology, and Challenges, Mason [/bib_ref]. However, these radioisotopes are costly and not readily available worldwide. To help overcome these limitations, researchers have developed various IONP nanoprobes that can detect Aβ plaques in the brain via a conjugated Aβ antibody on MRI in transgenic AD mice [bib_ref] SPION-enhanced Magnetic Resonance Imaging of Alzheimer's Disease Plaques in AβPP/PS-1 Transgenic Mouse..., Sillerud [/bib_ref] [bib_ref] Towards Non-invasive Diagnostic Imaging of Early-Stage Alzheimer's Disease, Viola [/bib_ref] [bib_ref] Non-invasive Screening for Early Alzheimer's Disease Diagnosis by a, Li [/bib_ref]. Nanotechnology-based PET has also been used in preclinical models to detect Aβ plaques in vivo [bib_ref] Coordination Chemistry of Bifunctional Chemical Agents Designed for Applications in 64Cu PET..., Sharma [/bib_ref] , demonstrating the potential for use in diagnostic imaging of human AD patients. ## Ii) parkinson's disease Progressive degeneration of dopaminergic neurons in the basal ganglia substantia nigra with abnormal deposition of αsynuclein protein is thought to cause the bradykinesia, and dystonia seen in PD patients . Aggregates of α-synuclein seen in other nuclei in the brain are thought to further contribute to the non-motor symptoms seen in PD patients such as hyposmia and dysautonomia, making αsynuclein an attractive biomarker for tracking disease Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 855195 2 progression [bib_ref] Selective Neuronal Vulnerability in Parkinson Disease, Surmeier [/bib_ref]. 123 I-ioflupane labelled dopamine SPECT can be used to monitor olfactory nerve dysfunction and serial MRI of basal ganglia are current tools for diagnosis and tracking of PD [bib_ref] The Clinical Benefit of Imaging Striatal Dopamine Transporters with [123I]FP-CIT SPET in..., Booij [/bib_ref] [bib_ref] EANM Practice Guideline/SNMMI Procedure Standard for Dopaminergic Imaging in Parkinsonian Syndromes 1.0, Morbelli [/bib_ref]. Novel non-invasive preclinical imaging tools to detect fibrillary α-synuclein currently lack adequate sensitivity and specificity. A recent study using a dLight1 fluorescence sensor to detect dopaminergic signals in the brains of transgenic PD mice yielded promising preliminary results in being able to detect fluorescent signals in dopaminergic neurons [bib_ref] Ultrafast Neuronal Imaging of Dopamine Dynamics with Designed Genetically Encoded Sensors, Patriarchi [/bib_ref] , while a dopamine-responsive nanoprobe that reacts in the near-infrared spectrum detected dopamine signals in preclinical models of drug abuse and PD [bib_ref] Near-Infrared Fluorescent Nanoprobes for Revealing the Role of Dopamine in Drug Addiction, Feng [/bib_ref] , suggesting that these experimental imaging modalities and targeted nanoprobes could have potential applications in human diagnosis of PD. ## Nanotechnology for the imaging of cerebral ischemia Acute blockage of blood vessels in the brain leads to cerebral ischemia or stroke, which requires prompt diagnosis and treatment to prevent permanent ischemic injury to the areas of the brain supplied by the affected blood vessels to prevent motor or sensory deficits [bib_ref] The Science of Stroke: Mechanisms in Search of Treatments, Moskowitz [/bib_ref]. Ischemic stroke is the second most common cause of death worldwide after cardiovascular disease, with approximately 87% mortality if left untreated. The ability to detect and locate the blockage and visualize the affected region of the brain with high sensitivity and spatial resolution can allow for accurate diagnosis, treatment, and serial monitoring of the treatment outcomes. MRI and MR perfusion scans to assess cerebral blood flow are the current gold standard non-invasive modalities for stroke imaging. Similar to the use of NPs to augment contrast enhancement for the detection of brain tumor vasculature, researchers have used NPs to increase the sensitivity and cellular resolution of areas of the brain affected by ischemia. IONPs conjugated with RGD peptide targeting the αvβ3 integrin receptors on endothelium have been used to enhance imaging of collateral vessels that form in the surrounding area of ischemia [bib_ref] Timely Visualization of the Collaterals Formed during Acute Ischemic Stroke with Fe3..., Wang [/bib_ref]. Another study comparing carbohydrate wrapped ferumoxytol to a Gdbased contrast agent found that ferumoxytol was able to detect cerebral blood volume at higher resolution at steady state than dynamic susceptibility contrast MR perfusion imaging using Gd [bib_ref] High-resolution Steady-State Cerebral Blood Volume Maps in Patients with Central Nervous System..., Varallyay [/bib_ref]. By leveraging the relative acidic environment in the ischemic penumbra, pH-responsive IONPs encapsulated in poly(β-amino ester)-poly(aminodoamine)poly(ethylene glycol) block copolymer were released and accumulated in the ischemic region of the brain in a rat model of cerebral ischemia, enabling detection on MRI [bib_ref] pH-Responsive Polymeric Micelle Based on PEG-Poly(β-Amino Ester)/(amido Amine) as Intelligent Vehicle for..., Gao [/bib_ref]. A safety study of IONPs in canines and macaque monkeys demonstrated sensitive detection of ischemia on T1weighted MR sequences in cerebral ischemia-induced animals, providing further preclinical evidence supporting their safety for use in human stroke patients [bib_ref] Iron Oxide Nanoclusters for T 1 Magnetic Resonance Imaging of Nonhuman Primates, Lu [/bib_ref]. CT angiography (CTA) using iodinated contrast agent provides 3-dimensional reconstructed images of large vessel occlusion. Encapsulation of CT contrast agents may facilitate enhanced delivery of imaging agents to the affected region of the brain. Polyethylene glycol (PEG) wrapped or PEGylated BaHoF 5 nanoprobes with an average diameter of 7 nm exhibited superior enhancement properties compared to the clinical contrast agent iohexol ® in CTA and CT perfusion imaging of a rat model of left middle cerebral artery occlusion . Gold NPs have also been used due to their biocompatibility and ability to incorporate targeting, therapeutic, and imaging moieties for multimodal imaging and theranostics. Chitosan-coated gold NPs with the ability to target fibrin were used to quantitate the amount of thrombolysis in a cerebral thromboembolic model of stroke in mice following treatment with intravenous tissue plasminogen activator (tPA). Serial CT imaging detected rapid thrombolysis following tPA treatment [bib_ref] Quantitative Imaging of Cerebral Thromboemboli In Vivo, Kim [/bib_ref]. Ultrasonography can be used to detect and image flow through blood vessels. Ultrasound contrast enhancers such as gas-filled microbubbles increase the "echogenicity" of the vessels, thereby increasing their chances to be detected using ultrasound [bib_ref] Microbubble Compositions, Properties and Biomedical Applications, Sirsi [/bib_ref]. These microbubbles are composed of albumin, lipids, or polymers and can be filled with air, perfluorocarbons, or nitrogen, and can be functionalized with surface ligands for targeting [bib_ref] Evolution of Contrast Agents for Ultrasound Imaging and Ultrasound-Mediated Drug Delivery, Paefgen [/bib_ref]. Perfluorocarbon emulsions conjugated with anti-fibrin antibodies have demonstrated the ability to visualize thrombus in a model of cerebral ischemia on ultrasound . IONPs have also been used to visualize areas of relative signal attenuation using microwave imaging in a rabbit model of cerebral ischemia as well as in a human volunteer [bib_ref] Iron Nanoparticle Contrast Enhanced Microwave Imaging for Emergent Stroke: A Pilot Study, Hudson [/bib_ref]. Applying a PEG coating to the surface of IONPs can greatly increase the circulation time of these NPs in the bloodstream , enabling threedimensional angiography following a single injection of PEGylated IONPs to allow for real-time imaging of cerebral vasculature in mouse models to assess cerebral hemorrhage in a mouse model of traumatic brain injury over a period of hours [bib_ref] X-space MPI: Magnetic Nanoparticles for Safe Medical Imaging, Goodwill [/bib_ref] [bib_ref] Firstin Vivotraumatic Brain Injury Imaging via Magnetic Particle Imaging, Orendorff [/bib_ref]. Taken together, these emerging uses of NPs to enhance multimodal imaging and assessment of cerebral ischemia may hopefully translate into the clinic to improve patient outcomes. Please refer to an excellent review on the emerging uses of nanotechnology in the imaging of stroke by Kaviarasi and colleagues for further information on this topic [bib_ref] Emerging Paradigms in Nanotechnology for Imaging and Treatment of Cerebral Ischemia, Kaviarasi [/bib_ref] and for ease of reference for our readers, we have compiled a list of NPs that are in human clinical trials or have been approved for neurological disorders . # Discussion Implementation of nanoscale materials for neuroimaging is still in the early stages of preclinical development. Several limitations exist that currently prevent the widespread application of nanomaterials for use in the CNS space. The presence of the intact BBB with endothelial tight junctions that limit passage of most materials greater than 100 nm in diameter restricts the bulkiness of nanomaterials that can be delivered systemically into the brain [bib_ref] Physiologic Upper Limit of Pore Size in the Blood-Tumor Barrier of Malignant..., Sarin [/bib_ref]. The cellular complexity of the CNS makes it difficult to target NPs to specific cells of interest (i.e. neurons and not glia) and avoid unintended off-target effects to different types of nearby cells. Finally, the brain is bathed in cerebrospinal fluid (CSF), which pulses with each heartbeat, creating a natural washout effect that actively prevents accumulation of cargo at the desired site of delivery. Furthermore, even with targeted delivery approaches using functionalized NPs that preferentially recognize protein receptors on cell surfaces (i.e., transferrin receptors on the surfaces of glioma cells), the percent injected dose of NPs that ultimately cross the BBB via the bloodstream is meager and would require dosing human subjects with hundreds of milliliters of NPs intravenously to achieve a detectable phenotypic effect [bib_ref] Analysis of Nanoparticle Delivery to Tumours, Wilhelm [/bib_ref]. More invasive mechanisms of bypassing the BBB such as direct administration of NPs into the brain using stereotactic implantable catheters or use of convection enhanced delivery or local ultrasonic disruption of the BBB can improve the uptake of NPs, but these tools are limited to specialized hospital centers, require collaborative efforts from a neurosurgical team, and are generally not recommended for use in day-to-day neuroimaging procedures [bib_ref] Temporary Disruption of the Blood-Brain Barrier by Use of Ultrasound and Microbubbles:..., Mcdannold [/bib_ref] [bib_ref] Ommaya Reservoir Insertion: A Technical Note, Magill [/bib_ref]. Metallic NPs are currently the most researched nanomaterials for emerging neuroimaging technologies. Due to their physicochemical compositions, metal oxide NPs have been incorporated in a broad range of biomedical applications, from imaging contrast agents to scaffolding materials for tissue regeneration. Their small size (~50 nm) also makes them ideal for trafficking across the BBB into the CNS space to enhance neuroimaging modalities and deliver various types of cargo for the treatment of neurological disorders [bib_ref] Superparamagnetic Iron Oxide: Clinical Application as a Contrast Agent for MR Imaging..., Stark [/bib_ref] [bib_ref] Physical and Chemical Properties of Superparamagnetic Iron Oxide MR Contrast Agents: Ferumoxides,..., Jung [/bib_ref] [bib_ref] Combined Iron Oxide Nanoparticle Ferumoxytol and Gadolinium Contrast Enhanced MRI Define Glioblastoma..., Barajas [/bib_ref]. The iron oxide contrast agent ferumoxytol (Feraheme ® , AMAG) has seen increasing usage in MR imaging in North America, while the IONP formulation ferumoxtran-10 (Sinerem/Combidex) has been gaining interest in human clinical trials in Europe [bib_ref] Noninvasive Detection of Clinically Occult Lymph-Node Metastases in Prostate Cancer, Harisinghani [/bib_ref] [bib_ref] MRI with a Lymph-node-specific Contrast Agent as an Alternative to CT Scan..., Heesakkers [/bib_ref] [bib_ref] Ultrasmall Superparamagnetic Particles of Iron Oxide Allow for the Detection of Metastases..., Triantafyllou [/bib_ref] [bib_ref] Noninvasive Differentiation between Hepatic Steatosis and Steatohepatitis with MR Imaging Enhanced with..., Smits [/bib_ref]. With decades of longitudinal data regarding the biodistribution, pharmacokinetics, and pharmacodynamics of IONPs, we now have a deeper understanding of how IONPs behave in the CNS and its associated neurotoxicities, which may limit their 1 | Nanoparticles approved or currently in clinical trials for neurological diseases. List of nanoparticles that are in human clinical trials or have been approved for the treatment of neuro-oncologic, neurodegenerative, and cerebovascular disorders. ## Neuro-oncology Trade name Product formulation Level of clinical development and treatment applications SGT-53 [bib_ref] Nanoparticles in the Clinic, Anselmo [/bib_ref] Transferrin-functionalized liposomal plasmid wild-type p53 DNA NCT02340156-Phase II open label, single-arm, multicenter study of IV SGT-53 and oral temozolomide in patients with confirmed recurrent glioblastoma or progression. DepoCyt [bib_ref] Revolutionary Impact of Nanodrug Delivery on Neuroscience, Khanbabaie [/bib_ref] Liposomal cytarabine Approved for treatment of malignant lymphomatous meningitis. DaunoXome [bib_ref] Revolutionary Impact of Nanodrug Delivery on Neuroscience, Khanbabaie [/bib_ref] Liposomal daunorubicin Approved for HIV-related Kaposi's sarcoma. Neulasta [bib_ref] Pros and Cons of the Liposome Platform in Cancer Drug Targeting, Gabizon [/bib_ref] [bib_ref] Innovative Pharmaceutical Development Based on Unique Properties of Nanoscale Delivery Formulation, Kumar [/bib_ref] PEGylated granulocyte colony-stimulating factor Approved for treatment of chemotherapy-associated neutropenia. Cornell Dots [bib_ref] Nanoparticles in the Clinic, Anselmo [/bib_ref] PEGylated silica NPs of 124 I-cRGDY NIR fluorophore Phase 0 safety trial of imaging of melanoma and malignant brain tumors. ## Neurodegenerative disorders Trade Name Product Formulation ## Level of clinical development and treatment applications Visudyne [bib_ref] Revolutionary Impact of Nanodrug Delivery on Neuroscience, Khanbabaie [/bib_ref] Liposomal verteporfin Approved for treatment of age-related mascular degeneration, pathologic myopia, and ocular histoplasmosis. Macugen [bib_ref] Pros and Cons of the Liposome Platform in Cancer Drug Targeting, Gabizon [/bib_ref] [bib_ref] Innovative Pharmaceutical Development Based on Unique Properties of Nanoscale Delivery Formulation, Kumar [/bib_ref] PEGylated anti-VEGF aptimer Approved for age-related macular degeneration. Partisiran [bib_ref] Nanoparticles in the Clinic, Anselmo [/bib_ref] Liposomal transthyretin siRNA Phase I-III study for the treatment of hereditary transthyretin-mediated amyloidosis. Characterized by peripheral sensorimotor and autonomic neuropathy. RNA interference that reduces production of both wild-type and mutant transthyretin protein. Phase III APOLLO study showed significant improvement in polyneuropathy. Copaxone [bib_ref] Pros and Cons of the Liposome Platform in Cancer Drug Targeting, Gabizon [/bib_ref] [bib_ref] Innovative Pharmaceutical Development Based on Unique Properties of Nanoscale Delivery Formulation, Kumar [/bib_ref] Copolymer of L-Glutamic acid, L-alanine, and L-tyrosine Approved for the treatment of Multiple Sclerosis. ## Cerebrovascular disorders ## Trade name product formulation level of clinical development and treatment applications Definity [bib_ref] Nanoparticles in the Clinic, Anselmo [/bib_ref] P e r flutren lipid microspheres Approved ultrasound contrast agent for imaging of cerebral ischemia and transcranial brain injuries. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org July 2022 | Volume 10 | Article 855195 translational potential. In vitro studies have demonstrated uptake of IONPs into neurons, glia, and astrocytes via micropinocytosis and clathrin-mediated endocytosis with trafficking into lysosomal compartments [bib_ref] Endocytotic Uptake of Iron Oxide Nanoparticles by Cultured Brain Microglial Cells, Luther [/bib_ref] [bib_ref] Internalization of Titanium Dioxide Nanoparticles by Glial Cells Is Given at Short..., Huerta-García [/bib_ref] [bib_ref] Surface Coating Affects Uptake of Silver Nanoparticles in Neural Stem Cells, Pongrac [/bib_ref]. Exposure to silver NPs in a BBB co-culture model led to shrinkage of mitochondria, expansion of the endoplasmic reticulum, and vacuolation in astrocytes with altered gene expression across 23 genes associated with metabolic, biosynthetic, and cell death processes [bib_ref] Silver Nanoparticles (AgNPs) Cause Degeneration of Cytoskeleton and Disrupt Synaptic Machinery of..., Xu [/bib_ref]. Astrocytes increased production of reactive oxygen species and decreased viability when exposed to copper oxide NPs [bib_ref] Uptake and Toxicity of Copper Oxide Nanoparticles in Cultured Primary Brain Astrocytes, Bulcke [/bib_ref]. Interestingly, studies have shown that the toxicity of IONPs depended on the surface coating. Polydimethyloamine coating caused cell membrane disruption and cell death in cultured cortical neurons, while aminosilane coating left the cell membrane intact but affected cellular metabolism, but dextran remained inert to cultured neurons [bib_ref] Altering Iron Oxide Nanoparticle Surface Properties Induce Cortical Neuron Cytotoxicity, Rivet [/bib_ref]. In vivo toxicity experiments have demonstrated accumulation of aluminum NPs in mouse brain endothelial cells causing neurovascular damage and increased BBB permeability , while acute exposure of gold NPs to male Wistar rats led to catabolic and energy metabolic suppression in the hippocampus, striatum, and cerebral cortex but long-term exposure only resulted in inhibition of catalase and energy metabolism in the cortex [bib_ref] Effect of Acute and Long-Term Administration of Gold Nanoparticles on Biochemical Parameters..., Ferreira [/bib_ref]. Taken together, these data indicate that neurotoxicity studies need to be done in higher order organisms such as non-human primates to better understand the mechanisms of neurotoxicity associated with NPs that are applied for CNS applications in humans. We recently reviewed emerging applications of nanotechnology in the fields of neurosurgery, neuro-oncology, and neuroradiology from the perspective of clinician-scientists [bib_ref] Integrating Nanotechnology in Neurosurgery, Neuroradiology, and Neuro-Oncology Practice-The Clinicians, Lam [/bib_ref]. One emerging area is the ability to imbue NPs with theranostic capabilities-functionalizing NPs with the dual ability to detect cells of interest and deliver therapeutic cargo. A recent first-in-human trial using the EGFR antibody cetuximab conjugated to the near-infrared (NIR) fluorescent dye IRDye800 (cetuximab-IRDye800) during neurosurgical resection of gliomas demonstrated the specificity of the antibody-dye conjugate to specifically attach to EGFR-expressing glioma cells in the brain, enhancing the detection of tumor cells during surgery and potentially assisting the neurosurgeon in achieving a more extensive surgical resection, which could improve patient survival. Conjugation of a CD133 monoclonal antibody to a phototoxic phthalocyanine NIR dye IR700 allowed for specific targeting and imaging of CD133+ glioma stem cells and treatment using NIR photoimmunotherapy to cause tumor shrinkage and cell death in a murine intracranial orthotopic model of glioma [bib_ref] Imaging and Selective Elimination of Glioblastoma Stem Cells with Theranostic Near-Infrared-Labeled CD133-specific..., Jing [/bib_ref]. Multi-walled carbon nanotubes have been used to replace conventional silver-silver chloride electrodes used in electroencephalography to eliminate the metal-associated artifacts seen in CT and x-ray images of the brain, to allow for better visualization of the brain during image-guided neurosurgical procedures [bib_ref] Thin-film Electroencephalographic Electrodes Using Multi-Walled Carbon Nanotubes Are Effective for Neurosurgery, Awara [/bib_ref]. Nanoknives made from silicon nitride with 20 nm tips can make minute and precise incisions to reduce the amount of cerebral tissue damage associated with using conventional neurosurgical tools [bib_ref] In Vivo use of a Nanoknife for Axon Microsurgery, Chang [/bib_ref]. Finally, dextrancoated IONPs covalently linked to tPA in an agarose gel can enhance the fibrinolytic activity of tPA for days, prolonging thrombolysis and potentially improving outcomes for stroke victims [bib_ref] Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for..., Heid [/bib_ref]. With further development of these exciting preclinical discoveries, there is hope that researchers will be able to bridge the gap from benchtop to bedside. # Conclusion The application of nanotechnology for use in neuroimaging has gained steady progress. Although this platform is still in its early stages of technology development, the ability to harness NPs as a theranostic will further enhance the function of NPs in the CNS to treat a wide variety of neurological disorders. The ability to decrease the neurotoxic nature of NPs will improve their safety profile and improve the translational potential for use in patients. # Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

The APOE ε4 exerts differential effects on familial and other subtypes of Alzheimer's disease Introduction: The genetic risk effects of apolipoprotein E (APOE) on familial Alzheimer's disease (FAD) with or without gene mutations, sporadic AD (SAD), and normal controls (NC) remain unclear in the Chinese population. Methods: In total, 15 119 subjects, including 311 FAD patients without PSEN1, PSEN2, APP, TREM2, and SORL1 pathogenic mutations (FAD [unknown]); 126 FAD patients with PSENs/APP mutations (FAD [PSENs/APP]); 7234 SAD patients; and 7448 NC were enrolled. The risk effects of APOE ε4 were analyzed across groups. Results: The prevalence of the APOE ε4 genotype in FAD (unknown), FAD (PSENs/APP), SAD, and NC groups was 56.27%, 26.19%, 36.23%, and 19.54%, respectively. Further, the APOE ε4 positive genotype had predictive power for FAD (unknown) risk (odds ratio: 4.51, 95% confidence interval: 3.57-5.45, P < .001). Discussion: APOE ε4 positive genotype may cause familial aggregation, and the investigation of multiple interventions targeting APOE pathological function to reduce the risk for this disease warrants attention.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. # Introduction The aging population in China has reached an unprecedented level. Dementia, especially Alzheimer's disease (AD), has become a serious social and family burden. [bib_ref] Dementia in China: epidemiology, clinical management, and research advances, Jia [/bib_ref] AD is classified into familial Alzheimer's disease (FAD) and sporadic Alzheimer's disease (SAD). FAD is almost entirely genetically determined, with heritability ranging from 92% to 100%. [bib_ref] Autosomal recessive causes likely in early-onset Alzheimer disease, Wingo [/bib_ref] It is characterized by an early age of onset and pedigree clustering. FAD has been widely researched over the years since its first identification. The pathogenic mutations in the amyloid precursor protein (APP), [bib_ref] Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease, Sherrington [/bib_ref] presenilin 1 (PSEN1), [bib_ref] Candidate gene for the chromosome 1 familial Alzheimer's disease locus, Levy-Lahad [/bib_ref] and presenilin 2 (PSEN2) [bib_ref] Segregation of a missense mutation in the amyloid precursor protein gene with..., Goate [/bib_ref] genes involved in the amyloid beta (Aβ) peptide processing, leads to development of FAD. However, these mutations underlie FAD in only a small proportion of cases, leaving a large group of familial subtypes genetically unexplained. [bib_ref] The patterns of inheritance in early-onset dementia: Alzheimer's disease and frontotemporal dementia, Jarmolowicz [/bib_ref] Advances in sequencing techniques have enabled the identification of rare mutations and variants with moderate-tostrong risk effects on this complex disease. Recently, next-generation sequencing studies have identified new loci such as sortilin-related receptor 1 (SORL1) [bib_ref] Genetic meta-analysis of diagnosed Alzheimer's disease identifies new risk loci and implicates..., Kunkle [/bib_ref] and triggering receptor expressed on myeloid cells 2 (TREM2), [bib_ref] TREM2 variants in Alzheimer's disease, Guerreiro [/bib_ref] suggesting the role of functional pathways other than Aβ processing in AD pathogenesis. These new genes and mechanistic pathways could inspire new diagnostic concepts or therapeutic targets for AD. Apolipoprotein E (APOE) is regarded as the greatest risk gene for AD; among the three isoform alleles (ε2, ε3, and ε4), APOE ε4 is considered the primary genetic risk factor. [bib_ref] Apolipoprotein e and Alzheimer disease: Risk, mechanisms and therapy, Liu [/bib_ref] Homozygous APOE ε4 carriers usually exhibit earlier onset of the disease than heterozygous carriers, highlighting the dose-effect of the ε4 allele on AD. One copy of the ε4 allele increases the risk of AD by ≈2 to 6 times, and the presence of two copies increases the risk by 7.2 to 21.8 times (African Americans 7.2, Hispanics 8.6, white 11.8, Japanese 21.8). [bib_ref] Apolipoprotein e and Alzheimer disease: Risk, mechanisms and therapy, Liu [/bib_ref] [bib_ref] Apolipoprotein E in Alzheimer's disease and other neurological disorders, Verghese [/bib_ref] [bib_ref] Effects of age, sex, and ethnicity on the association between apolipoprotein E..., Farrer [/bib_ref] [bib_ref] Systematic meta-analyses of Alzheimer disease genetic association studies: the AlzGene database, Bertram [/bib_ref] [bib_ref] Association of apolipoprotein E genotype and Alzheimer disease in African Americans, Murrell [/bib_ref] [bib_ref] Local ancestry at APOE modifies Alzheimer's disease risk in Caribbean Hispanics, Blue [/bib_ref] The general frequency of the APOE ε4 allele ranges from 9% to 23% in diverse ethnic populations (Asian 9%, Hispanic 12%, white 14%, African descent 19%, other/mixed 23%), but dramatically increases in AD patients (Hispanic 24%, Asian 28%, African descent 35%, white 38%, other/mixed 45%).The frequency of the APOE ε4 allele also varies among these subtypes. [bib_ref] Apolipoprotein E ε4 and age at onset of sporadic and familial Alzheimer..., Olarte [/bib_ref] A recent study in a cohort of 404 Chinese subjects with FAD showed that among patients without PSENs/APP mutations, [bib_ref] Genomics: new light on Alzheimer's disease research, Jung [/bib_ref].31% carried one APOE ε4 allele, while 14.85% carried two APOE ε4 alleles.This suggests that APOE ε4 plays a major role in cases of FAD without PSENs/APP mutations. These results challenge the role of APOE as a risk factor mainly for SAD development. Therefore, a large-scale, multicenter study is necessary to investigate the effects of APOE in FAD, especially in those without PSENs/APP mutations. Our aim was to explore the distribution and genetic effects of the APOE ε4 genotype in FAD without APP, PSEN1, PSEN2, TREM2, and SORL1 mutations. In addition, we compared the frequency of APOE in the Chinese population with data available from other countries. The outcomes of this study provide more clarity regarding the regulation of AD by the APOE ε4 positive genotype. # Methods ## Participants In ## Procedures The and CDR sum of boxes (SOB) scores (range 0-18) were recorded. The following four groups were included in this study: FAD the National Institute on Aging-Alzheimer's Association criteria.The detailed inclusion and exclusion criteria are provided in in supporting information. Blood samples were drawn by venipuncture at baseline and DNA was extracted by salting-out procedures, as previously described. [bib_ref] A simple salting out procedure for extracting DNA from human nucleated cells, Miller [/bib_ref] The Sanger sequencing method was used to determine the APOE genotypes. Exon 4 of the APOE gene was amplified by PCR using the follow- ## Statistical analyses Descriptive statistics were used to summarize the participant char- subjects showed that the genetic risk effect of the APOE ε4 positive genotype differed across subtypes of AD. Among these, the FAD (unknown) was the most affected. We propose that the APOE ε4 positive genotype may cause familial aggregation; therefore, the APOE ε4 gene plays a major role in this clinical phenomenon. # Results ## Demographics The [bib_ref] Prevalence of Apolipoprotein E4 genotype and homozygotes (APOE e4/4) among patients diagnosed..., Ward [/bib_ref] [bib_ref] Association between DCHS2 gene and mild cognitive impairment and Alzheimer's disease in..., Vieira [/bib_ref] China is positioned at the lowest prevalence level. C, The prevalence of AD in the population in France is presented the highest (12%), followed by the United States (9.7%), Spain (8.5%), Japan (7.2%), Australia (6.7%), UK (6.5%), South Korea (5.2%), Germany (3.06%), Canada (3%), China (3%), Brazil (2.7%), and Colombia (1.8%) [bib_ref] Trends in dementia prevalence, incidence, and survival rate in a Japanese community, Ohara [/bib_ref] [bib_ref] Trends in prevalence of dementia in French farmers from two epidemiological cohorts, Pérès [/bib_ref] [bib_ref] Alzheimer's disease and vascular dementia in developing countries: prevalence, management, and risk..., Kalaria [/bib_ref] [bib_ref] Prevalence of dementia and subtypes in Valladolid, northwestern Spain: the DEMINVALL study, Tola-Arribas [/bib_ref] [bib_ref] Simultaneous temporal trends in dementia incidence and prevalence, 2005-2013: A population-based retrospective..., Kosteniuk [/bib_ref] [bib_ref] Short-term trends in dementia prevalence in Germany between the years, Doblhammer [/bib_ref] those in the FAD (PSENs/APP) group. The average years of education in each group ranged from 7 to 12 years. The differences in the MMSE, CDR, and CDR SOB scores among the various groups are presented in . ## Apoe allele frequencies across the diagnostic groups The . ## Apoe ε4 positive genotype carriers across the diagnosed groups The prevalence of the APOE ε4 in SAD patients in China was the lowest among 12 countries ; [bib_ref] Prevalence of Apolipoprotein E4 genotype and homozygotes (APOE e4/4) among patients diagnosed..., Ward [/bib_ref] [bib_ref] Association between DCHS2 gene and mild cognitive impairment and Alzheimer's disease in..., Vieira [/bib_ref] however, the prevalence of SAD in China was not the lowest . [bib_ref] Trends in dementia prevalence, incidence, and survival rate in a Japanese community, Ohara [/bib_ref]. Interestingly, the prevalence of the APOE ε4 positive genotype in the FAD (unknown) group was higher than that in the SAD or FAD (PSENs/APP) groups (P < .001), while the prevalence of the APOE ε4 positive genotype in the SAD group was higher than that in the FAD (PSENs/APP) group (P = .02). The APOE genotypes were classified as APOE ε4 heterozygous or homozygous, and their distribution in each group is shown in ,D. ## Predictive effect of apoe ε4 in ad To explore the predictive effect of APOE in the different AD subtypes, we used binary logistic regression models adjusted for age, sex, education, and region of subjects . We also examined the effect of APOE ε4 gene dosage on disease risk, and found that the OR values for two APOE ε4 copies (homozygous) were higher than those for a single copy (heterozygous) in the FAD (unknown) and SAD groups; this was especially evident in the FAD . ## F i g u r e 2 The prevalence and predictive effect of apolipoprotein E (APOE) genotypes. The radar charts show the prevalence of APOE genotypes in Alzheimer's disease (AD) subtypes and in normal population (A) and the odds ratio (OR) of APOE genotypes in AD subtypes (B). The prevalence and OR value of the APOE ε4/ε4 genotype was higher in the FAD (unknown) group compared to that in other groups. The bar graphs with the trendline indicates the rising frequencies and OR value in APOE ε4 heterozygous groups (C). The rising frequencies and OR in APOE ε4 homozygous genotypes in the FAD (unknown) group were also significantly higher than that in other groups (the OR value was presented with 95% confidence interval) (D). FAD (unknown), familial Alzheimer's disease without PSEN1, PSEN2, APP, TREM2, and SORL1 mutations; FAD, familial Alzheimer's disease; NC, normal control ## Risk effect of apoe genotype for ad across different ages in the fad (unknown) and sad groups We estimated the OR values of the APOE ε4 positive genotype in comparison with that for the APOE ε4 negative genotype across different ages in the FAD (unknown) groups and SAD group by binary logistic regression models adjusted for sex and education [fig_ref] 3: Future direction [/fig_ref]. We found that, in the same age range, the OR value increased with the number of APOE ε4 alleles in both groups. Moreover, we found that the risk due to the ε4 allele in both groups was age-dependent. In the FAD (unknown) group, the risk due to the ε4 allele gradually increased with age, and decreased gradually after 70 to 79 years of age. Similarly, in the SAD group, the risk for AD due to the ε4 allele gradually increased with age, and decreased after 65 to 74 years of age. ## Risk prediction probability model of fad (unknown) Our results using the logistic regression model showed that APOE genotype and age were significant risk factors for FAD (unknown). We calculated the risk scores based on different age ranges and the number of APOE ε4 copies. The total risk score range was 0 to 15 points, and each score corresponded to a risk prediction probability score; these TA B L E 3 Logistic regression analysis for predictive effect of APOE on AD subtypes # Discussion To our knowledge, this is the first multicenter study to examine APOE in the Chinese population and involved 15 119 individuals. We described the prevalence of the APOE alleles and genotypes in FAD (unknown), FAD (PSENs/APP), SAD, and NC groups and compared the genetic effects of APOE ε4 among these groups. We found that the APOE ε4 positive genotype increased the risk of AD, enhanced the familial aggregation, and showed a peak risk effect between 70 and 79 years of age in FAD (unknown) patients. Additionally, we found that the prevalence of the APOE ε4 positive genotype was clearly lower among the SAD patients in China than among those in Europe and the United States, indicating that ethnic background is an important factor in the risk of AD development. [bib_ref] Prevalence of Apolipoprotein E4 genotype and homozygotes (APOE e4/4) among patients diagnosed..., Ward [/bib_ref] [bib_ref] Association between DCHS2 gene and mild cognitive impairment and Alzheimer's disease in..., Vieira [/bib_ref] The prevalence of the APOE ε4 positive genotype displayed the group. These values were ≈2-fold higher than those reported in previous studies. [bib_ref] Apolipoprotein e and Alzheimer disease: Risk, mechanisms and therapy, Liu [/bib_ref] [bib_ref] Apolipoprotein E in Alzheimer's disease and other neurological disorders, Verghese [/bib_ref] [bib_ref] Effects of age, sex, and ethnicity on the association between apolipoprotein E..., Farrer [/bib_ref] Thus, based on our study, we propose that patients ## F i g u r e 3 The odds ratio (OR) and risk probability for Alzheimer's disease (AD) development among different apolipoprotein E (APOE) ε4 genotypes with different age ranges. In the FAD (unknown) group (A), and SAD group (B), the odds ratio (OR) value for developing AD was higher in the APOE ε4 +/+ group than in the APOE ε4 +/group, irrespective of the age range. In the FAD (unknown) group, the OR value increased with age, and decreased gradually after 70 to 79 years of age. In the SAD group, similar to FAD (unknown), the risk effect of the ε4 allele for AD gradually increased till 65 to 74 years of age and decreased thereafter (the OR value was presented with 95% confidence interval). C, The heat maps show the risk probability of FAD (unknown Our study had some inherent limitations. First, the number of patients with FAD was relatively small owing to its low incidence compared to SAD. Another reason is that the life span for FAD patients is usually shorter, and as such, we managed to enroll few patients over 70 years, which affects our result regarding the effects of age. We plan to expand this sample size in our future research. Second, although we believe that APOE ε4 may be a pathogenic gene with an incomplete penetrance in FAD (unknown), we did not perform functional in vitro or in vivo studies to verify this hypothesis in the FAD subtype. Third, we found that APOE ε4 had different genetic effects in different AD subtypes; however, this finding should be validated in longitudinal cohorts in the future. Fourth, although our model has capacity to predict the risk of FAD (unknown), there would be other factors such as family history that contribute to the development of FAD (unknown). Therefore, we should include more factors to improve the prediction efficiency in the future. Fifth, previous studies have identified some risk genes for AD, such as APOE, BIN1, CD33, EPHA1, SORL1, TOMM40 and so on. [bib_ref] Association of Polygenic Risk Score with Age at Onset and Cerebrospinal Fluid..., Li [/bib_ref] [bib_ref] Genomics: new light on Alzheimer's disease research, Jung [/bib_ref] [bib_ref] Genomics of Alzheimer's disease: value of highthroughput genomic technologies to dissect its..., Tosto [/bib_ref] We only tested APP, PSEN1, PSEN2, TREM2, and SORL1 mutations in our study and did not test the deletion/duplication, which may lead to the loss of genetic information. We will include this content in future research. # Conclusions This is the largest multicenter study investigating the association between APOE and AD in the Chinese population. This study revealed that the APOE ε4 positive genotype was associated with different AD subtypes and showed an increasing trend in NC, FAD (PSENs/APP), SAD, and FAD (unknown) groups, in that order. The high frequency of APOE ε4 in FAD (unknown) suggests that it plays an important role in familial aggregation. Future studies to identify therapeutic strategies for FAD (unknown) subtype should consider the age and APOE ε4 genotype. This might lead to identification of potential viable therapeutic options for patients in the FAD (unknown) family. # Acknowledgments We thank all participants in this study and all neurologists at relevant academic centers for their help in the recruitment of subjects. In particular, we thank the participating hospitals including The First [fig] 3: Future direction: Future research in this field should aim to elucidate the mechanisms by which the APOE ε4 positive genotype exerts genetic risk effects on familial clustering of the FAD (unknown) subtype. In addition, studies should seek to identify therapeutic agents targeting the pathological function of APOE ε4 in order to develop viable therapeutic options for patients with FAD (unknown). ε4or ε2positive genotype status on the cases (FAD [unknown], FAD [PSENs/APP], and SAD) compared to the NC after adjusting for age, sex, education, and region of subjects. We further calculated the risk of APOE genotype for AD across different ages in the FAD (unknown) group and SAD group using a binary logistic regression model after adjusting for sex and education. According to the Framingham risk scoring method described by Sullivan et al., 25 we established a FAD (unknown) prediction model. The FAD (unknown) risk prediction model is based on a binary logistic regression model, and APOE genotype, age, sex, and education were included as risk factors. The disease risk score was estimated according to the regression coefficient of the significant risk factors, and the risk heat map was drawn according to the risk prediction probability. The prediction accuracy of FAD (unknown) risk prediction model was estimated by the area under curve (AUC) using a logistic regression model. All statistical analyses were performed using SPSS version 22.0 (SPSS Inc, Chicago, IL, USA). P < .05 was considered to indicate significant results. [/fig]

A functional genetic screen identifies the Mediator complex as essential for SSX2-induced senescence # Introduction Cellular senescence is an aging-related growth arrest of cells that contributes to the pathology of many diseases of the elderly, such as diabetes, cardiovascular disease, and cancer 1,2 . Senescence was first described as a response to cell division-related telomere erosion in human diploid cells (i.e., replicative senescence), but over the years different types of cellular stress have been demonstrated to induce senescence, including DNA damage, oxidative stress, oncogene activation, and certain chemotherapeutic drugs [bib_ref] The hallmarks of aging, Lopez-Otin [/bib_ref] [bib_ref] Senescence in tumours: evidence from mice and humans, Collado [/bib_ref]. Although much has been learned about the initiating factors of senescence and the phenotype of senescent cells, the molecular basis of the senescence response is still largely uncharacterized. Since the economic and social burdens associated with demographic shifts in many countries have prompted the need for better diagnostics and treatment of age-related diseases, we need a better understanding of the senescence response. In cancer, senescence may act as a powerful barrier to cellular transformation by blocking the effects of activated oncogenes. This is supported by in vivo observations of nevi, which invariably express activated oncogenes together with classical senescence-associated markers (e.g., senescence-associated beta-galactosidase activity and p16 activation) [bib_ref] The Mediator complex: a master coordinator of transcription and cell lineage development, Yin [/bib_ref] [bib_ref] Improving classification of melanocytic nevi: Association of BRAF V600E expression with distinct..., Kiuru [/bib_ref] [bib_ref] Genetics of melanocytic nevi, Roh [/bib_ref] [bib_ref] High frequency of BRAF mutations in nevi, Pollock [/bib_ref] [bib_ref] BRAFE600-associated senescence-like cell cycle arrest of human naevi, Michaloglou [/bib_ref]. Furthermore, nevi show no signs of telomere attrition, suggesting that the senescence response is driven by oncogene expression rather than loss of replicative potential 7 . Oncogene-induced senescence has been studied in several in vitro models with strong oncogenes such as Ras, B-Raf, Cyclin E, and Myc, which suggest that replication stress may be instrumental for development of this phenotype We recently demonstrated that SSX2 induces senescence in different types of cells, as determined by classical senescence features, including enlargement of the cytoplasm, cell growth arrest, enhanced B-galactosidase activity, and DNA double strand breaks [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. SSX proteins are germ cell-specific proteins that are expressed at the spermatogonial stage of spermatogenesis [bib_ref] Cancer-germline antigen vaccines and epigenetic enhancers: future strategies for cancer treatment, Gjerstorff [/bib_ref]. They are also ectopically expressed in many types of tumors, such as 40% of melanomas and up to 65% of breast cancers [bib_ref] Prospective study on the expression of cancer testis genes and antibody responses..., Mischo [/bib_ref] [bib_ref] SSX2-4 expression in early-stage non-small cell lung cancer, Greve [/bib_ref]. The SSX family comprise nine highly identical members, which are most likely redundant in their cellular functions [bib_ref] Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins, Traynor [/bib_ref]. SSX proteins belong to the cancer/testis (CT) antigen group of tumor antigens, and T-cell and antibody responses have been detected in cancer patients [bib_ref] Oncogenic cancer/testis antigens: prime candidates for immunotherapy, Gjerstorff [/bib_ref]. The role of SSX proteins in tumor formation and progression remains largely elusive. We have demonstrated that knockdown of SSX genes in melanoma cancer cells significantly reduces cellular proliferation [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref] and SSX proteins were shown to activate several important mitogenic pathways, such as MAPK and Wnt [bib_ref] Oncogenic functions of the cancer-testis antigen SSX on the proliferation, survival, and..., D&apos;arcy [/bib_ref]. The role of SSX proteins in supporting cell proliferation and their ability to induce senescence suggest that these CT antigens may have oncogenic potential. SSX proteins are DNA-binding factors that regulate chromatin structure and Polycomb function [bib_ref] Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins, Traynor [/bib_ref] [bib_ref] Oncogenic cancer/testis antigens: prime candidates for immunotherapy, Gjerstorff [/bib_ref] [bib_ref] SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation..., Gjerstorff [/bib_ref] [bib_ref] Epigenetic reprogramming of pericentromeric satellite DNA in premalignant and malignant lesions, Bruckmann [/bib_ref]. They contain an SSX repression (SSXRD) domain and a Kruppel-associated box (KRAB), which seem to be important for chromatin-binding and -rearrangement, respectively 14 . However, the specific mechanism by which SSX proteins regulate chromatin structure remains unknown. In this study, we performed a functional genetic screen to identify factors essential for SSX2-induced senescence in order to further understand the molecular functions of SSX2 and the cellular response to SSX2. The results showed that several subunits of the Mediator complex are important for SSX2-induced senescence. The Mediator complex is directly implicated in RNA polymerase IImediated transcription, and its main function is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery [bib_ref] The mediator complex: at the nexus of RNA polymerase II transcription, Jeronimo [/bib_ref]. Thus, Mediator is important for integration of signals from different signaling pathways that impinge on chromatin to regulate gene transcription. In addition, the Mediator complex seems to play a role in chromatin structure and the spatial positioning of chromatin in the nucleus. It has been demonstrated that Mediator interacts with the SWI/SNF and CHD1 chromatin remodeling complexes [bib_ref] SWI/SNF-dependent chromatin remodeling of RNR3 requires TAF(II)s and the general transcription machinery, Sharma [/bib_ref] [bib_ref] Mediator coordinates PIC assembly with recruitment of CHD1, Lin [/bib_ref] [bib_ref] Targeting of Swi/Snf to the yeast GAL1 UAS G requires the Mediator,..., Lemieux [/bib_ref] and is important for heterochromatin formation [bib_ref] Mediator directs co-transcriptional heterochromatin assembly by RNA interference-dependent and -independent pathways, Oya [/bib_ref]. Mediator is also important for the formation and stabilization of chromatin loops [bib_ref] Mediator and cohesin connect gene expression and chromatin architecture, Kagey [/bib_ref] [bib_ref] Architectural protein subclasses shape 3D organization of genomes during lineage commitment, Phillips-Cremins [/bib_ref] [bib_ref] Nipbl and mediator cooperatively regulate gene expression to control limb development, Muto [/bib_ref] and for regulation of gene expression by association with the nuclear pore complex [bib_ref] The nuclear pore-associated TREX-2 complex employs mediator to regulate gene expression, Schneider [/bib_ref]. The human Mediator complex generally consists of 26 subunits, but the complex seems highly flexible in structure [bib_ref] The Mediator complex: a central integrator of transcription, Allen [/bib_ref]. Several studies have indicated that subsets of endogenous Mediator complexes may lack specific subunits and therefore acquire different functions. For instance, the thyroid hormone receptor binds the complex through MED1, and MED1 knockout cells have impaired activation of hormone receptor target genes 29 , but Mediator complexes lacking MED1 could integrate signals from transcription factors that bind other subunits. In addition, the Mediator complex associates with additional factors, such as the four subunit CDK8 module, which dramatically alters its structure and function [bib_ref] The Mediator complex: a central integrator of transcription, Allen [/bib_ref]. These results suggest that the transcriptional integration of different signaling events and associated transcription factors rely on specific components and possible compositions of the Mediator complex. In accordance, Mediator complexes with a simplified subunit composition have been identified in differentiated cells [bib_ref] MyoD targets TAF3/TRF3 to activate myogenin transcription, Deato [/bib_ref]. Mediator subunits have been shown to exhibit cancer-specific transcription profiles that indicate disease-related shifts in Mediator complex composition [bib_ref] Comprehensive analysis of the transcriptional profile of the Mediator complex across human..., Syring [/bib_ref]. Furthermore, different subunits have been implicated in cancer development or progression 32 . As described above, the Mediator complex is important in multiple cellular functions and there is growing evidence for a role in cancer. In this study, we describe for the first time a role for Mediator in SSX2-mediated senescence. # Results A functional genetic screen identified potential mediators of SSX2-induced senescence Functional genetic screening using shRNA libraries is a powerful tool for identification of genes essential for specific cellular phenotypes. We applied this technique to a previously established model 10 , wherein MFC7 cells with ectopic expression of SSX2 , b) undergo a senescence response, evident by loss of proliferation, enhanced beta-galactosidase activity, and increased cell size . MCF7 cells were transduced with lentivirus carrying an shRNA library targeting 4922 human genes with 4-6 shRNAs per gene . Since SSX proteins are DNA-binding factors with a role in structural regulation of chromatin and might induce senescence through a chromatin-associated mechanism, we selected a library constructed by Cellecta that was focused towards genes encoding DNA binding molecules. Following lentiviral infection with shRNA-encoding plasmid constructs, the cells were treated with doxycycline to induce SSX2 expression. Subsequently, the cells were cultured for 21 days to allow senescence development (approximately 7 days) and outgrowth of cell clones that did not undergo senescence-associated cell growth arrest due to shRNA-mediated knockdown of specific genes. Cells without SSX2 expression were cultured in parallel to control for genes that affect MCF7 cell growth in general. Massive parallel sequencing of the baseline sample (MCF7 cells infected with the library), post-senescence samples and non-senescence control samples in two replicates identified a list of shRNAs enriched in post-senescence samples that might target genes essential for the development of SSX2-induced senescence . There was a high concordance between the two replicates . Eleven genes that were targeted by at least two shRNAs enriched more than fivefold in the post-senescence samples, compared to baseline samples and the non-senescence samples, were considered potential mediators of SSX-induced senescence . The presence of several shRNAs targeting SSX2 and homologous SSX genes was indicative of a successful approach . ## Mediator complex subunits are essential for ssx2-induced senescence Among the 11 genes identified, the most prominent were subunits of the Mediator complex ; Supplementary Tables 1 and 2). Mediator complex subunits 1 and 4 (MED1 and MED4) were represented by three and two shRNAs, respectively, but single shRNAs targeting MED9, MED14, MED21, MED22, MED23, and MED26 were also highly enriched in post senescence samples . This strongly suggested that the Mediator complex is important for SSX2-induced senescence. To validate this, we targeted the expression of MED1, MED4, and MED14 by novel sets of shRNAs in a single geneknockdown format. Despite testing many different shRNAs targeting MED1, MED4, and MED14, a general knockdown efficiency of only approximately 50-70% suggested that complete knockdown of these genes was not tolerated by the cells, which is in accordance with the central role of the Mediator complex in Functional genetic screening for identification of genes essential for SSX2-induced senescence. a-e MCF7 cells undergo senescence in response to doxycycline-inducible expression of SSX2. This cell model has been established previously [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. Expression of SSX2 was validated by Western blotting (a) and immunocytochemical staining (b). For Western blot, Coomassie staining of the membrane was included as loading control. After 7 days of culture with doxycycline, the cells exhibited loss of proliferation as determined by a crystal violet staining assay. Stained cells were solubilized and staining levels were quantified at OD570 (c). Proliferation assays were done with EdU incorporation for 24 h (d). The cells also acquired a morphology typical for senescent cells (i.e., enlarged cell size and cytoplasm) and exhibited beta-galactosidase activity (e). f MCF7 cells with doxycycline-regulated SSX2 were transduced with a pooled shRNA library containing 27,500 shRNA targeting 4922 genes and cultures with or without doxycycline for 21 days. The frequency of individual shRNAs was quantified at baseline, after establishment of SSX2-induced senescence and in a non-senescence growth control, using next generation sequencing (NGS). g shRNAs enriched in doxycycline-treated samples (two replicates) compared to untreated samples and baseline were identified that might target genes essential for SSX2-induced senescence. SSX and Mediator shRNAs enriched more than fivefold in doxycycline (DOX)-treated samples, compared to untreated and baseline samples are highlighted. As expected, SSX shRNAs were identified together with shRNAs targeting genes encoding multiple subunits of the Mediator complex. Data represents the mean ± SD for three biological replicates. ***< 0.0001. Scale bars = 25 µm Reduced levels of Mediator subunits prevents SSX2-mediated senescence. a MCF7 cells with doxycycline (DOX)-inducible SSX2 expression were transduced with plasmids encoding shRNAs specific for genes encoding the Mediator subunits MED1, MED4, or MED14. Relative expression levels of MED1, MED4, and MED14 were determined using quantitative RT-PCR. b-e MCF7 cells with MED1, MED4, or MED14 shRNAmediated knockdown were treated with doxycycline to induce SSX2 expression and cultured for 7 days. Cell growth was then measured using a crystal violet cell staining followed by quantification at OD570 (b). Representative pictures are shown (c). The frequencies of senescent cells were identified by beta-galactosidase assay (d) and quantified (e). f HEK293 cells were previously shown to acquire a senescence-like morphology and growth arrest in response to doxycycline-inducible expression of SSX2 [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. Expression of SSX2 was validated by Western blotting. Coomassie staining of the membrane was included as loading control. g We also knocked down MED1 expression in this model and determined the efficiency by quantitative RT-PCR. h The effect of MED1 knockdown on cell growth was evaluated 7 days after induction of SSX2 expression by crystal violet cell staining followed by quantification at OD570. i The expression levels of MED1, MED4, and MED14 were quantified by RT-PCR in MCF7 cells with and without doxycycline-induced SSX2 expression. Data represent the mean ± SD for three biological replicates. ns = non significant. ***< 0.0001; **< 0.001; *< 0.01. Scale bars = 100 µm. a and g show the average of three technical replicates therefore no standard deviations are shown transcriptional regulation. However, reduced levels did not seem to perturb cell growth . Interestingly, this reduction in expression level was sufficient for the MCF7 cells to bypass SSX2-induced, senescence-associated growth arrest and confirmed the role of MED1, MED4, and MED14 in this phenotype . In addition, a strong reduction in the frequency of enlarged and senescence-associated, beta-galactosidase-positive cells was observed following knockdown of MED1 expression. To investigate whether the role of the Mediator complex in SSX2-induced senescence was cell typespecific, we knocked down MED1 with shRNAs in the HEK293 cell line model with inducible SSX2 expression [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. HEK293 cells have been demonstrated to be prone to undergo senescence [bib_ref] RAC3 more than a nuclear receptor coactivator: a key inhibitor of senescence..., Fernandez Larrosa [/bib_ref]. As for MCF7 cells, the level of MED1 expression was reduced to about 50% and reduced levels of MED1 facilitated bypassing SSX-induced senescence-associated growth arrest , suggesting a more general role of the Mediator complex in SSX2-induced senescence. The expression levels of individual Mediator Complex subunits were not changed during SSX2-induced senescence . We also tested the ability of SSX2 to induce senescence in IMR90 fibroblast cells, which can undergo other types of senescence. SSX2 reduced the growth of the cells, but did not induce senescence characteristics such as enlarged cell size and beta-galactosidase activity . ## Med1 is important for ssx2-mediated activation of p21 transcription In a previous study, we showed that SSX2-induced senescence was associated with increased levels of p53 and its downstream target, the cycline-dependent kinase inhibitor p21 [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. There is strong evidence that p53 activity and the p53-p21 axis are major regulators of senescence in human cells [bib_ref] Four faces of cellular senescence, Rodier [/bib_ref]. Thus we investigated the role of p53 and two other important tumor suppressor proteins, Rb and PTEN, in SSX2-mediated senescence in MFC7 cells . SSX2 expression was associated with increased levels of p53 and its downstream target p21 . Rb was slightly reduced a later time points (4 and 7 days), while PTEN remained unchanged . Knockdown of TP53, RB1, and PTEN confirmed the importance of p53 in SSX2-mediated senescence . Thus we investigated whether bypass of the SSX2-induced senescence response by knockdown of MED1 expression would reduce levels of p53 and p21. Indeed, we found that MCF7 cells with shRNA-mediated knockdown of MED1 levels during induction of SSX2 expression exhibited reduced levels of both p53 and p21 . Interestingly, SSX2 expression did not enhance mRNA levels of TP53 (encoding p53), suggesting that increased protein levels were achieved through protein stabilization . The role of MED1 in SSX2-induced activation of the p53-p21 axis was further indicated by reduced CDKN1A (encoding p21) expression in MCF7 cells with SSX2 expression and MED1 knockdown . Mediator complex subunits are not essential for the senescence response in general Having demonstrated a role for the Mediator complex in SSX2-mediated senescence in MCF7 and HEK293 cells, we investigated its role in SSX2-independent senescence using a model of H-Ras oncogene-induced senescence in MCF7 cells [bib_ref] Dissecting the senescence-like program in tumor cells activated by Ras signaling, Bihani [/bib_ref]. Following overexpression of H-Ras G12V in MCF7 cells [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref] , a senescent phenotype and growth arrest similar to SSX2-expressing MCF7 cells was observed [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. Next, we investigated whether knockdown of MED1 expression in this model diminished senescence development in response to H-Ras expression. Interestingly, no significant effect of MED1 knockdown on senescence-associated growth inhibition or morphological changes induced by H-Ras was observed [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref] , suggesting that the Mediator complex is important for the cellular response to SSX2, but not to H-Ras. We also investigated the role of Mediator in a druginduced senescence model, where MCF7 cells undergo senescence in response to brief Epirubicin treatment [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. Knockdown of MED1 in this model did not affect the Epirubicin-induced increase in betagalactosidase activity and increase in cell size [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. Nor did it significantly reduce senescence-associated loss of proliferation, although there was a tendency towards such an effect [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. These results suggest a more specific role for the Mediator complex in the molecular function exerted by SSX2 in MCF7 cells, which ultimately leads to senescence. It could also be due to mechanistic differences in the senescence response to SSX2 and H-Ras/Epirubicin, where only the former may depend on the conserved activity of the Mediator complex. ## Expression of mediator subunits is deregulated in melanoma Our results showing that reduced expression of MED1, MED4, and MED14 prevented SSX2-mediated senescence suggests that diminished Mediator complex activity might be a compensating factor for maintaining proliferation in response to SSX expression in tumors. To this end, we investigated the potential deregulation of the Mediator complex subunits in clinical tumor samples. A direct correlation between the Mediator complex activity and SSX expression is complicated by the many different Mediator complex subunits and SSX members, and thus decreased activity of the Mediator complex in a particular tumor could be facilitated by reduced expression of any of the subunits. In agreement, there was no obvious anti-correlation between expression of SSX2 and different Mediator subunits in tumors . Thus, instead of a direct correlation of SSX2 and Mediator complex levels, we investigated the potential deregulation of Mediator complex subunits in various cancer types, including breast cancer and melanoma, which often express SSX2 and other SSX proteins [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. In general, TCGA RNA-seq data showed that expression of MED1, MED4, and MED14 are not lost in tumors, but varies considerably between tumor specimens compared normal [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. In some cancer types, the expression was reduced in tumors compared to normal tissues, but in others it was increased. In breast cancer, MED1 and MED4 expression was significantly reduced in tumors, while MED14 was increased [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. In melanoma, MED1 and MED14 was increased, while MED4 was reduced [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. Strikingly, MED1 expresssion was significantly enhanced in HER2positive breast cancers, while reduced in the basal-like subtype [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. MED1 and MED4 expression was generally slightly reduced with breast cancer progression [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref] , while MED14 expression was generally increased [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. We also investigated the expression of MED1 using immunohistochemical staining. First, we validated the specificity of the MED1 antibody by staining a panel of normal tissues. As expected, that MED1 was detected in all tissues except the liver, and was localized predominantly in the nuclei [fig_ref] Figure 6: The level of MED1 changes during melanoma tumorigenesis [/fig_ref] , in accordance with data from the Human Protein Atlas (www.proteinatlas.org). Next, we investigated if MED1 expression is deregulated in association with loss of the senescence response during tumorigenesis. This was most appropriately done in melanoma, where premalignant specimens are easily accessible (i.e., nevi). Thus, we quantified MED1 levels in a panel of benign nevi (n = 39) and melanoma tumors (n = 74) using immunohistochemical staining. Melan-A The effect of MED1 in SSX2-mediated activation of p53-p21. a The SSX2-mediated induction of important tumor suppressors was investigated using Western blotting at indicated time points after addition of doxycycline to induce SSX2 expression. Coomassie staining of the membrane was included as loading control. b The expression of genes encoding p53, Rb, and PTEN was knocked down using lentiviral transductions with shRNA expression plasmids and confirmed with Western blotting. Coomassie staining of the membrane was included as loading control. c The effect of p53, Rb, and PTEN knockdown on SSX2-mediated growth arrest was investigated using crystal violet staining aften 6 days of doxycyclineinduced SSX2 expression and quantified at OD570. d The effect of 6 days of doxycycline-induced SSX2 expression on the levels of p53 and p21 in MCF7 cells with and without reduced MED1 expression was investigated using Western blotting. Coomassie staining of the membrane was included as loading control. e, f The expression levels of TP53 (e) and CDKN1A (f) were quantified by RT-PCR in MCF7 cells with and without doxycyclineinduced SSX2 expression. *< 0.01 was stained on parallel tissue sections for identification of melanocytic lesions (for an example see [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref]. The analysis demonstrated consistent medium levels of MED1 expression in the nuclei of all melanocytic cells of benign dermal and compound nevi, whereas expression in the nuclei of melanomas was much more inconsistent, including specimens with high, medium, or low/no expression [fig_ref] Figure 6: The level of MED1 changes during melanoma tumorigenesis [/fig_ref]. Within both benign and malignant tumors, MED1 exhibited a relatively homogenous expression pattern [fig_ref] Figure 6: The level of MED1 changes during melanoma tumorigenesis [/fig_ref]. Immunohistochemical staining of MED1 showed that 31% and 27% of melanoma specimens exhibited high or low/no expression, respectively [fig_ref] Figure 6: The level of MED1 changes during melanoma tumorigenesis [/fig_ref]. These results were consistent with a previous study reporting that MED1 was often either upregulated or downregulated in melanoma [bib_ref] Down-regulation of the expression of RB18A/MED1, a cofactor of transcription, triggers strong..., Ndong Jde [/bib_ref] , which also showed that low MED1 expression correlated with a highly tumorigenic phenotype. Multiple other studies have also demonstrated that MED1 31,37-41 and Mediator subunits are deregulated in melanoma and other cancer types [bib_ref] Comprehensive analysis of the transcriptional profile of the Mediator complex across human..., Syring [/bib_ref] [bib_ref] Expression of Med19 in bladder cancer tissues and its role on bladder..., Zhang [/bib_ref] [bib_ref] Amplification and expression of genes from the 17q11 approximately q12 amplicon in..., Luoh [/bib_ref] [bib_ref] Comprehensive copy number and gene expression profiling of the 17q23 amplicon in..., Monni [/bib_ref] [bib_ref] Elevated MED28 expression predicts poor outcome in women with breast cancer, Yoon [/bib_ref] [bib_ref] Med19 promotes gastric cancer progression and cellular growth, Ding [/bib_ref]. The observed differences in expression of different MEDs may translate into diffences in the activity of the Mediator complex to support tumorigenesis and specific phenotypes, such as SSX2 expression. # Discussion The SSX2 protein, like well-established oncogenes such as B-Raf, Ras, Cyclin D, and Myc, supports cancer cell proliferation, but at the same time induces senescenceassociated growth arrest when ectopically expressed [bib_ref] Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic..., Greve [/bib_ref]. This paradox most likely reflects the need for additional alterations that prevent or bypass the senescence response. Identifying such alterations is an important step toward a better understanding of tumor initiation. In this study, we used a functional genetic screening approach to identify genes that, when reduced in expression, prevent a SSX2-induced senescence response. Interestingly, we identified several subunits of the Mediator complex as essential for this phenotype to develop. . d-f Reduced levels of MED1 does not prevent enhanced betagalactosidase activity (d), the acquisition of senescent morphology (e), or senescence-associated growth arrest (f) in epirubicin treated cells. Cells were analyzed 7 days after treatment. Data represent the mean ± SD for three biological replicates. ns = non significant. Scale bars = 100 µm Notably, it was only possible to achieve 50-80% knockdown of MED1, MED4, and MED14, and cells with reduced expression exhibited reduced growth. This suggested that complete loss of Mediator was not tolerated by the cells, which is in accordance with other studies showing that complete knockout of Mediator subunits is embryonically lethal and impairs cell growth and cell cycle progression 3 , but in disagreement with another study showing that complete MED1 knockdown is indeed possible [bib_ref] Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer..., Cui [/bib_ref]. However, a reduction in the levels of Mediator subunits was enough to prevent SSX2-induced senescence in MCF7 and HEK293 cells. Interestingly, SSX2 did not induced a senescence response in IMR90 fibroblast cells, suggesting that that the ability of SSX2 to induce senescence is dependent on cell types or specific molecular patterns. The Mediator complex has a multifaceted role in integrating cellular signals into changes into transcriptional reprogramming, but has not been described in association with senescence. Senescence is a complex phenotype, which, in addition to loss of proliferative signaling, also involves major changes to the chromatin regulation, metabolism and structure of the cell [bib_ref] The Mediator complex: a central integrator of transcription, Allen [/bib_ref]. A role for Mediator in senescence development was, therefore, not unexpected. Moreover, the Mediator complex has previously been linked to the function of p53. In one study, Mediator subunits, including cofactor CDK8, were shown to be recruited to the CDKN1A locus together with p53 and modulate the strength of p53-mediated transcriptional activation [bib_ref] CDK8 is a stimulus-specific positive coregulator of p53 target genes, Donner [/bib_ref]. In another study, p53 was demonstrated to activate transcription by directly binding and changing the conformation of the Mediator complex [bib_ref] p53 activates transcription by directing structural shifts in Mediator, Meyer [/bib_ref]. Thus, there seems to be an intimate relationship between p53-activity and the functions of the Mediator complex. Whether interactions with the Mediator complex also stabilizes p53, as indicated here , remains to be determined. The prominent role of p53 in the senescence response and the direct link between the Mediator complex and p53 activity further suggested a role for the Mediator complex in senescence. However, knockdown of MED1 in other models of senescence in which MCF7 cells undergo senescence in response to the H-Ras oncogene or Epirubicin suggested that the activity of the Mediator complex per se was not instrumental for development of senescence [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. Our data linked the Mediator complex to the SSX-mediated senescence. There are several posibile ways that the Mediator complex may mediate SSX2-induced senescence. The Mediator complex may be central for the senescence response to SSX2 perhaps by regulating the gene expression of central factors. This does not seem to include regulation of TP53 as this gene was not significantly induced in response to SSX2 expression. The Mediator complex may also be important for SSX2 functions, which include but may not be limited to, chromatin regulation/rearrangement. The latter is supported by our recent data demonstrating that SSX proteins bind and rearrange chromatin in cancer cells, and it is likely that the effect of the SSX2 protein on chromatin structure and gene expression is what initiates SSX2mediated senescence. Importantly, SSX proteins seem to play an antagonistic role in Polycomb group (PcG) gene repression, suggesting that ectopic expression of SSX molecules may release PcG-repressed genes 17 and facilitate binding of RNA polymerase II preinitiation complex (PIC) to change the transcription landscape. In agreement with this, PcG complexes have been demonstrated to directly inhibit the assembly of the PIC 50 . Thus, the Mediator complex may facilitate the SSX-mediated changes in gene expression that initiates a senescence response. Another possibility is that the SSX2-mediated structural rearrangement of chromatin involves the Mediator complex, which is important for several aspects of chromatin organization. An important implication of our results is that reduced levels of MED1, MED4, or MED14 may support specific cancer-associated alterations (e.g., SSX2 expression). This observation is interesting in the light that the expression of multiple subunits of the Mediator complex, including MED1, MED4, or MED14, is frequently dysregulated in different types of cancer. # Materials and methods Cells and culture conditions MCF7-SSX2, HEK293-SSX2 cells were grown in DMEM (Sigma Aldrich, Brondby, Denmark), 10% fetal bovine serum (FBS) and penicillin/streptomycin, supplemented with 6 ng/ml insulin (MCF7). When relevant, cell identities according to ATCC were verified using DNA fingerprinting by short tandem repeat analysis (Cell IDTM system, Promega) and tested for mycoplasma (MycoAlert, Mycoplasma detection kit, Lonza). MCF7 cells were were treated for two hours with 1 µM Epirubicin to induce senescence. IMR90 cells were cultured in EMEM (Sigma Aldrich, Brondby, Denmark), 10% FBS and penicillin/streptomycin. IMR90 cells were transduced with a lentiviral vector for expression of doxycyclineinducible SSX2 expression as previously described [bib_ref] Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins, Traynor [/bib_ref]. shRNA library preparation The shRNA screen was conducted using the Decipher module 3 pooled shRNA library (Cellecta Inc.), which contains 27500 shRNAs targeting 4922 human genes (i.e., 5-6 shRNAs per gene). The library was prepared as lentiviral particles by co-transfection of HEK293T cells with second-generation packaging plasmids psPAX2 and pMD2.G (Cellecta Inc.) using Lentifectin transfection reagent (Biocat, Germany). Lentiviral particles were harvested after 72 h and concentrated using Lenti-X concentrator (Takara Clontech). Functional genetic screening with a pooled shRNA library MCF7 cells with doxycycline-inducible expression of SSX2 (i.e., MCF7-SSX2) were infected with lentiviral particles carrying the pooled shRNA library in the presence of 5 µg/ml polybrene to obtain 40% transduced cells. Cell numbers were adjusted to maintain a library representation of >400. Cells were seeded in culture plates and selected with 0.2 µg/ml puromycin for 5 days. Cells were harvested and reseeded at a density of approximately 2.2 × 10 3 cells/cm 2 in four replicates. The remaining cells were frozen for later use as baseline samples. Two replicates were supplemented with 100 ng/ml doxycycline to induce SSX2 expression and cells were cultured for 21 days with addition of fresh media and doxycycline every three days. Next, gDNA was isolated from the baseline sample, no-doxycycline controls and doxycycline-treated cells using the Blood and cell culture maxi kit (Qiagen) according to the manufacturers recommendations and sheared into smaller fragments using a tip-sonicator set at 20% for 2 × 10 s. shRNA library barcodes were amplified with nested polymerase chain reaction (PCR) (see for primers) using Titanium Taq DNA polymerase (Clontech-Takara). For the first PCR, 200 µg of gDNA were used to maintain library coverage and reactions were run for 16 cycles. A fraction of the product (1/80) was reamplified with indexing primers for 14 cycles in a second PCR. Products were purified from a 3% agarose gel using the Nucleospin gel and PCR cleanup kit (Macherey-Nagel, Dueren, DE) and quantified with Quant-iT picogreen reagent (firma). Samples were pooled and subjected to end sequencing using a Hiseq sequencer (Illumina, San Diego, CA, USA). Sequencing data were aligned to the library of barcode identifiers using the Barcode deconvoluter software (Cellecta) and normalized to total reads. Only barcodes identified with more than 100 reads in the doxycyclinetreated samples were considered in the further analysis comparing barcode frequencies from the baseline sample, no-doxycycline controls and doxycycline-treated cells. ## Growth assays Cells were seeded in 6-well plates at a density of 5000 cells/well, and after 24 h 100 ng/ml of doxycycline was added. After approximately 10 days, plates were washed in PBS and stained with 5 mg/ml crystal violet in 25% methanol, H 2 O for 15 min. After 3 washes in H 2 O, plates were air-dried and photographed or the crystal violet stain was solubilized in buffer consisting of 29.41 mg/ml citrate in 50% ethanol, H 2 O and quantified on a Spectramax Paradigm reader (Molecular Devices) at OD570. ## Senescence-associated beta-galactosidase assay Senescence-associated beta-galactosidase activity was measured at pH 6.0 using a senescence beta-galactosidase staining kit (Cell Signaling) according to the manufacturer's recommendations. ## Western blotting Extracts were made from monolayers of cells with RIPA buffer, resolved by 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked in PBS, 0.1% Tween-20, and 5% non-fat dry milk powder and then incubated with anti-SSX2-4 (1:3000) [bib_ref] Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and..., Santos [/bib_ref] , p53 (Santa Cruz Biotechnology, sc-126, 1:500), p21 (Santa Cruz Biotechnology, sc-53870, 1:1000), Rb (Cell Signaling, #9309, 1:2000), PTEN (Cell Signaling, #9188, 1:1000), or anti-H-Ras (Santa Cruz Biotechnology, sc-520, 1:1000). The blot was further stained with horseradish peroxidaseconjugated goat anti-mouse IgG (DakoCytomation Denmark A/S, Glostrup, Denmark) and developed with an ECL Western Blot kit (Amersham Biosciences, Hilleroed, Denmark, 1:100.000). All antibody incubation and washing steps were carried out in PBS, 0.1% Tween-20. Quantitative RT-PCR RNA was purified from cells using RiboZol (VWR) followed by cDNA synthesis using the RevertAid Premium Reverse Transcriptase kit from Fermentas. Quantitative real-time PCR was performed using SYBR green based expression analysis (Applied Biosystems) in combination with Quantitect primers: MED1 #QT00076356, MED4 #QT00039158, MED14 #QT00063490, PUM1 #QT00029421, CDKN1A (#QT00062090), and TP53 (#QT00060235). ## Shrna knockdown Annealed oligoes were ligated into the pSico plasmid using standards methods. The shRNA plasmids were prepared as lentivirus by cotransfection with pMD2. G, pRSV-Rev, pMDL g/p RRE (kindly provided by the Trone Lab through Addgene, Cambridge, UK) into HEK293T cells using Lentifectin (Abmgood, Richmond, Canada). Virus was harvested from the supernatant after 2 and 3 days, filtered, precipitated with PEG and resuspended in PBS. For TP53, CDKN1A and PTEN knockdown, pLKO1 shRNA plasmids were purchased from Sigma Aldrich and prepared as lentivirus as described above. ## Indirect immunofluorescence Cells grown on coverslips were fixed in 4% formaldehyde, permeabilized in 0.2% Triton X100, PBS and blocked in 3% bovine serum albumin (BSA), PBS. Immunostaining was done with anti-SSX2/SSX3 (clone 1A4; Sigma Aldrich; 1:100) in 1% BSA, PBS, and goat antimouse IgG (H+L) cross-adsorbed Alexa Fluor 488/568 (Thermo Fisher Scientific). Cells were mounted under cover slides with ProLong Gold Antifade with DAPI (Life Technologies) and imaging was performed with an Olympus IX73 microscope fitted with a PlanApo N 60X/ 1.42 oil objective. ## Immunohistochemical staining Tissues sections were deparaffinized and treated with 1.5% H 2 O 2 in Tris-buffered saline (pH 7.5) for 10 min to block endogenous peroxidase activity. Tissues were then washed in TNT buffer (0.1 m Tris, 0.15 m NaCl, 0.05% Tween-20, pH 7.5) and subjected to antigen retrieval with different protocols, including microwave boiling for 15 minutes in (1) T-EG buffer (10 mm Tris, 0.5 mm EGTA, pH 9.0), (2) 10 mm citrate buffer (pH 6.0), or (3) Dako Target retrieval solution (Dako S1699), or proteolytic treatment using (4) 0.05% protease type XIV (pronase E, Sigma, cat. no. P5147) in TBS (pH 7.0) for 15 minutes at 37°C or (5) 0.4% pepsin (Sigma, Cat. No.: P7012) in 0.01 m HCl for 20 min at 37°C. Optimal atigen retrieval was achieved with microwave boiling in T-EG buffer for 15 min. Sections were then incubated with rabbit anti-TRAP220/MED1 antibody (NB100-2574; Novus Biologicals) or mouse anti-Melan A (Clone A103; Ventana Medical Systems) diluted in antibody diluent (S2022, DAKO Cytomation, Glostrup, Denmark) for 1 h at room temperature. Subsequently, sections were washed with TNT, incubated with EnVision Flex/HRP+ for 30 minutes, washed again and incubated with 3,3′diaminobenzidine (DAB)+ substrate-chromogen for 10 min. Following another wash with H 2 O, sections were counterstaining with Mayers hematoxylin before mounting in AquaTex (Merck Inc., Whitehouse Station, NJ, USA). Normal tissues analyzed for MED1 expression included: Tonsil, skin, esophagus, parotis, lung, thyroid, spleen, liver, gall bladder, colon, duodenum, muscle, testis, prostate, bladder, kidney, uterus, and placenta. ## Statistical testing Statistical testing was done using the t test to compared two groups or the one-way ANOVA for comparing multiples groups [fig_ref] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence [/fig_ref]. For statistical testing of larger data sets [fig_ref] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors [/fig_ref] , the Mann-Whitney U test was used. [fig] Figure 4: MED1 is not important for H-Ras oncogene-and epirubicin-induced senescence. a MCF7 cells stably transduced with MED1 shRNA expression vectors or empty vector control were transduced with an H-Ras G12V expression plasmid or empty control. H-Ras G12V expression was confirmed by Western blotting. Coomassie staining of the membrane was included as loading control. b, c In 3-4 days, the cells expressing H-Ras G12V acquired a senescent phenotype (b) and underwent growth arrest (c) [/fig] [fig] Figure 5: Expression of Mediator subunits in melanoma and breast cancer tumors. The expression levels of genes encoding the Mediator subunits MED1, MED4, and MED14 were examined in breast cancer and melanoma using RNA-seq data from the TCGA repository. Results are presented as relative expression levels (normalized number of reads). a Expression of MED1, MED4, and MED14 in normal breast epithelia and breast tumors (BRCA) and in normal skin and melanoma tumors (SKCM). b Expression of MED1, MED4, and MED14 indifferent subtypes and stages of breast cancer. Number of specimens are indicated. The p values were calculated using the (unpaired) Mann-Whitney U test [/fig] [fig] Figure 6: The level of MED1 changes during melanoma tumorigenesis. a The level of MED1 was examined using immunohistochemical staining in normal tissues. Medium levels of expression were observed in all tissues except the liver. Representative stainings are shown. b The level of MED1 was also investigated in 39 dermal and compound nevi and in 74 primary and metastatic melanoma tumors. The level of MED1 was quantified as high, medium, or low/negative. Representative pictures are shown. c Quantification of the results from A and B show consistent MED1 expression levels among nevi and diverging levels among tumors. Scale bars: A = 100 µM, B = 25 µm [/fig]

Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections Background: In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined. Methods: Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion. Results: The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%.Conclusion: This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa. # Background Despite significant reduction in malaria-related morbidity and mortality in the recent past, malaria remains endemic in the tropics and sub-tropics including sub-Saharan Africa. About 225 million clinical cases and 781,000 deaths were reported worldwide in 2009, whereby almost 90% occurred in sub-Saharan Africa. Current malaria control strategies include the use of insecticide-treated bed nets (ITNs), indoor residual spraying of insecticide, intermittent preventive treatment to young children and pregnant women, and early parasitological diagnosis and treatment of clinical cases using artemisinin-based combination therapy (ACT) [bib_ref] Defining and defeating the intolerable burden of malaria III. Progress and perspectives, Breman [/bib_ref] [bib_ref] Insecticidetreated bed-nets for malaria mosquito control, Curtis [/bib_ref]. Deployment of these strategies has had significant impact on malaria in many endemic areas. Although the impact of current malaria control strategies is encouraging, much more remains to be done to attain elimination. In the meantime, malaria vaccine is considered as an additional necessary arsenal. However, genetic diversity in Plasmodium falciparum is a major limitation for the successful development of an effective malaria vaccine, as it influences the level and efficacy of acquired protective immunity to malaria. Therefore, screening of the genetic diversity of malaria parasite populations in different endemic settings is an important step towards the development and/or the evaluation of malaria vaccines [bib_ref] A review of malaria vaccine clinical projects based on the WHO rainbow..., Schwartz [/bib_ref]. Genotyping of merozoite surface protein-2 (msp2) is the commonly used method to study the genetic diversity of P. falciparum. This gene presents polymorphisms in the number of repeats and sequence type [bib_ref] Age dependent carriage of multiple Plasmodium falciparum merozoite surface antigen-2 alleles in..., Ntoumi [/bib_ref]. The analysis of msp2 alleles involves two major allelic families, FC27 and 3D7. The msp2 gene is also the basis of determining the multiplicity of infections (MOI) in infected individuals, which is defined as the number of distinguishable P. falciparum clones per infected individual. MOI is a good indicator of acquired immunity or premunition of the populations living in endemic areas and is also correlated to the transmission intensity [bib_ref] Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region..., Babiker [/bib_ref] [bib_ref] Epidemiology of multiple Plasmodium falciparum infections. Age dependence of the multiplicity of..., Smith [/bib_ref]. It has been demonstrated that MOI could be influenced by parasite density and age [bib_ref] Migot-Nabias F: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal:..., Vafa [/bib_ref] , and haemoglobin (Hb) type carriage [bib_ref] Plasmodium falciparum: sickle-cell trait is associated with higher prevalence of multiple infections..., Ntoumi [/bib_ref]. Makélékélé is an administrative division in the southeastern part of Brazzaville (Republic of Congo), and has been well characterized as a highly endemic area with perennial malaria transmission [bib_ref] Criteria for diagnosing clinical malaria among a semi-immune population exposed to intense..., Trape [/bib_ref] [bib_ref] Malaria and urbanization in the central Africa: the example of Brazzaville. Part..., Trape [/bib_ref]. However, there is a paucity of information on the genetic diversity of P. falciparum populations. Following the replacement of chloroquine (CQ) with ACT (artesunate-amodiaquine and artemether-lumefantrine as first and second line treatment respectively) in 2006 for the treatment of uncomplicated malaria in the Republic of Congo, CQ drug pressure has since been reduced. In Malawi, absence of CQ pressure was followed by the re-emergence of CQ-susceptible malaria, based on genotyping of CQ resistance biomarker, P. falciparum chloroquine resistance transporter gene (pfcrt) [bib_ref] Re-emergence of chloroquinesensitive Plasmodium falciparum malaria after cessation of chloroquine use in..., Kublin [/bib_ref] [bib_ref] Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine..., Mita [/bib_ref] [bib_ref] Return of chloroquine antimalarial efficacy in Malawi, Laufer [/bib_ref]. In Congo Brazzaville, molecular analysis of the parasite population, including the CQ resistance pfct K76T mutation has not been investigated since the change of anti-malarial treatment policy in 2006. Therefore, the present study was conducted in the semi-urban area of Makélékélé to determine (i) the prevalence and the multiplicity of P. falciparum asymptomatic infection; (ii) the genetic diversity of the msp2 gene; and (iii) the prevalence of pfcrt K76T mutation in P. falciparum isolates. # Methods ## Study site The study conducted under the auspices of the Central Africa Network on Tuberculosis, HIV/AIDS and Malaria (CANTAM) project, was carried out in three districts of Makélékélé health division: Ngoko, Kinsana and Mbouono. These semi-urban divisions with about 6,000 inhabitants are located in the southern part of Brazzaville along the Congo River where malaria is transmitted throughout the year [bib_ref] Malaria and urbanization in the central Africa: the example of Brazzaville. Part..., Trape [/bib_ref]. Plasmodium falciparum is the main plasmodial species and Anopheles gambiae the main mosquito vector. ## Study population Children aged one to nine years and permanent residents of the study area were enrolled in a cohort for malaria surveillance study (Ndounga et al, unpublished). From October 2009 to June 2010, a demographic survey was conducted and 313 children under 10 years were enrolled. Malaria incidence is 0.9 malaria episode/year/ child. Inclusion criteria were absence of clinical malaria in the last two weeks and at least one week after enrolment, and with an axillary temperature of < 37.5°C. Recruitment was done from April to June 2010, and informed consent was obtained from parents or guardians. Thick and thin blood smears were made from each child. About 4 ml of whole blood were also collected in EDTA tube for haemoglobin concentration measurement and DNA extraction. This study was approved by the Institutional Ethics Committee for Research on Heath Sciences of the Republic of Congo. ## Microscopic examination Thick and thin blood films were stained with 10% Giemsa for 15 min and read by two independent competent microscopists to determine malaria species and the parasite density. Asexual parasites were counted against 200 leucocytes and expressed as the number of asexual parasites/μl of blood, assuming the leucocyte count of 8,000/μl of blood. ## Genomic dna extraction Genomic DNA was extracted from 200 μl of whole blood sample using the QIAmp DNA Blood Mini kit (QIAGEN Gmbh, Hilden, Germany) following the manufacturer's instruction. DNA was recovered in 200 μl of elution buffer provided in the kit and stored at -20°C until use. ## Msp2 genotyping of plasmodium falciparum isolates To characterize parasite infection, P. falciparum genotyping was performed using a nested PCR for msp2 central region, which comprises repeats of varying lengths. The FC27 and 3D7 allele families were analysed. The specific oligonucleotide primers and PCR conditions were as previously described by Ntoumi et al [bib_ref] Site-based study on polymorphism of Plasmodium falciparum MSP-1 and MSP-2 genes in..., Ntoumi [/bib_ref]. ## Pfcrt k76t pcr-typing The nested PCR followed by restriction enzyme digestion PCR reactions were used to genotype the Lysine to Threonine mutation in codon 76 of pfcrt as described by Mayengue et al [bib_ref] In vivo chloroquine resistance and prevalence of the pfcrt codon 76 mutation..., Mayengue [/bib_ref] , Djimde et al [bib_ref] Coulibaly D: A molecular marker for chloroquine-resistant falciparum malaria, Djimdé [/bib_ref] and Mayor et al [bib_ref] Prevalence of the K76T mutation in the putative Plasmodium falciparum chloroquine resistance..., Mayor [/bib_ref]. All isolates successfully amplified using msp2 PCR were assayed with the pfcrt PCR. Sickle cell trait β-globin genotypes HbAA, HbAS and HbSS were determined using allele specific PCR method as described by Wu et al [bib_ref] Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell..., Wu [/bib_ref]. # Data analysis The prevalence of FC27 and 3D7 alleles was determined as the presence of PCR products for each type in the total number of amplified bands for the corresponding locus. The MOI was determined as the number of different msp2 genotypes per isolate, and the mean MOI was calculated as the total number of detected Plasmodium falciparum msp2 genotypes/total number of infected children [bib_ref] In vivo chloroquine resistance and prevalence of the pfcrt codon 76 mutation..., Mayengue [/bib_ref]. The msp2 allelic frequency represents the ratio of one specific allele out of the total number of alleles identified in the isolates. The polyclonality (percentage of isolates with more than one amplified PCR fragment) was estimated for each group. Similarly, proportions of wild and mutant pfcrt alleles were calculated as the ratio of each allele type out of the total of P. falciparum positive samples. Statistical analysis was done using XLSTAT software (Version 2011.2.08). The chisquare test was used to compare quantitative variables such as parasite prevalence and MOI between different groups. The Spearman's rank correlation coefficient was calculated to assess association between MOI and parasite density. Differences were considered statistically significant at P values < 0.05. # Results ## Baseline demographic data and parasitological indexes A total of 313 children aged from one to nine years (mean age: 4.4 ± 2.5 years) were enrolled in the study. Sickle cell trait was present in 58 (18.5%) of them. The haemoglobin levels were distributed into 3 categories: normal ≥ 12.0 g/ dl (23.3% of children), moderate anemia 11.9 g/dl-7.0 g/dl (72.2%) and severe anemia < 7 g/dl (1.3%). The prevalence of asymptomatic parasitaemia detected microscopically was 8.6%, P. falciparum being the only infecting specie. The parasite density ranged from 62 to 16,090 parasites/μl and a geometric mean density of 3,421 parasites/μl. Through PCR amplification of msp2 gene, P. falciparum infection was detected in 16% of the isolates [fig_ref] Table 1: Demographic data and parasitological indexes of the study population [/fig_ref]. The prevalence of infection (by microscopy and PCR) varied according to the age of children (P < 0.05), being highest in the seven to nine years age group (14 and 26% respectively), followed by four to six years (11 and 17%), and one to three years age group (3.5 and 7%). ## Merozoite surface protein 2 genetic diversity Alleles were determined by size and family type. Allele typing showed the highly polymorphic nature of P. falciparum isolates. Both 3D7 and FC27 families were identified with 8 and 10 different alleles respectively [fig_ref] Figure 1: Distribution of P [/fig_ref]. The proportion of isolates having only 3D7 and FC27 alleles was 23/51 (45%) and 18/51 (35%) respectively. Both allelic families were found in 7/51 (14%) of the isolates. Six per cent (6%) of the isolates were not identified to any of the 3D7 or FC27 allelic family despite being repeatedly amplified [fig_ref] Table 2: Genetic diversity of P [/fig_ref]. This might be due to a mutation in the annealing region of the primers. ## Multiplicity of plasmodium falciparum infection In this study, 74% of the infections analysed consisted of a single msp2 genotype, 22% were double infection and 4% triple. The overall MOI was 1.3 and similar in all age groups. The proportion of samples with more than one clone was higher in both one to three and four to six years age groups (33.3 and 27.8% respectively) compared to the seven to nine years olds group (19%) [fig_ref] Table 3: Comparison of the parasite prevalence, parasite density and the MOI in different... [/fig_ref]. However, the difference was not statistically significant. ## Number of individuals 313 The haemoglobin phenotype (AA or AS) had no influence on MOI. The prevalence of multiple infections in AA group was 19% compared with 27% in AS group, and this difference was not significant [fig_ref] Table 3: Comparison of the parasite prevalence, parasite density and the MOI in different... [/fig_ref]. Nonetheless, a significant positive association was found between P. falciparum parasite density and MOI (Spearman rank coefficient = 0.4489, P = 0.0278) [fig_ref] Figure 2: Correlation of msp2 multiplicity of infection and mean Plasmodium falciparum parasite density... [/fig_ref]. ## Prevalence of pfcrt k76t mutation Genotyping of the pfcrt gene showed that 47 isolates out of 51 (92%) produced the expected 134 bp amplicon, suggesting the presence of T76 mutation. Four isolates (8%) with the wild type were found [fig_ref] Table 1: Demographic data and parasitological indexes of the study population [/fig_ref]. # Discussion In the Republic of Congo, less attention had been put on the investigation of the genetic diversity of P. falciparum among asymptomatic parasite carriers. Besides, this analysis is necessary to develop strategies for malaria control including the design of malaria vaccines interventions. The current study is the first of its kind to evaluate genetic diversity of P. falciparum isolates circulating among children younger than 10 years old, and to present the molecular analysis of pfcrt gene since the replacement of chloroquine with ACT as the first line antimalarial drug in Congo Brazzaville. The prevalence of P. falciparum carriage of 8.6% (microscopically) and 16% (by PCR) is low compared to those reported previously from other malaria endemic areas [bib_ref] Migot-Nabias F: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal:..., Vafa [/bib_ref] [bib_ref] Plasmodium falciparum: sickle-cell trait is associated with higher prevalence of multiple infections..., Ntoumi [/bib_ref] [bib_ref] Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from..., Cortés [/bib_ref]. However, these findings are in line with the results of a recent study conducted in Gabon that showed a relatively low prevalence of asymptomatic P. falciparum infection of 5% by microscopy among young children. In the recent past, a large number of ITNs were available to children under five years of age and to pregnant women in the Republic of Congo. The observed low prevalence could be attributed to the wide use of the ITNs. Further investigations involving more children and adults would be of great importance to confirm this finding. Allele-specific genotyping of msp2 has shown that P. falcuparum population in this part of the Republic of Congo have a high genetic diversity. Dispite the fact that a high proportion of isolates (74%) carried one No. Number a Total number of alleles is the number of different msp2 alleles distinguished by the size and the family type (3D7 and FC27) b Allelic frequency is defined as the ratio of 3D7 or FC27 alleles out of the total number of msp2 alleles genotype in comparison to those having two and three genotypes, different types of both 3D7 and FC27 alleles were found. The proportion of 3D7 alleles (55%) was slightly high compared with that of FC27 (45%). This is similar to findings in Papua New Guinea [bib_ref] Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from..., Cortés [/bib_ref]. The MOI (number of concurrent msp2 genotypes in individual infections) ranged from 1 to 3. The mean MOI of 1.3 is relatively low compared to those reported in Gabon [bib_ref] Human genetic polymorphisms and asymptomatic Plasmodium falciparum malaria in Gabonese schoolchildren, Mombo [/bib_ref] , Senegal [bib_ref] Migot-Nabias F: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal:..., Vafa [/bib_ref] [bib_ref] Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal, Jafari-Guemouri [/bib_ref] , and Ghana [bib_ref] Multiplicity of Plasmodium falciparum infection following intermittent preventive treatment in infants, Buchholz [/bib_ref] in asymptomatic cases. This might be explained by the fact that diversity of P. falciparum population differs according to the geographic location, and the level of transmission [bib_ref] Extensive dynamics of Plasmodium falciparum densities, stages and genotyping profiles, Färnert [/bib_ref] [bib_ref] Plasmodium falciparum population dynamics during the early phase of anti-malarial treatment in..., Carlsson [/bib_ref]. In the same study area [bib_ref] Genetic polymorphisms of merozoite surface protein-1 and surface protein-2 in Plasmodium falciparum..., Pembe [/bib_ref] , the MOI in isolates from symptomatic children was 2.2. The interpretation of this increase in the mean number of parasite genotype/infected child could be the presence of the uncontrolled strain causing the symptoms. Age is one of the suggested important factors involved in the acquisition of immunity against P. falciparum and influences MOI [bib_ref] Migot-Nabias F: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal:..., Vafa [/bib_ref]. On the contrary, these results show that age has no effect on MOI. The absence of influence of age on MOI has also been previously reported in the same area among P. falciparum symptomatic children by Pembe et al [bib_ref] Genetic polymorphisms of merozoite surface protein-1 and surface protein-2 in Plasmodium falciparum..., Pembe [/bib_ref]. Previous studies on association between MOI and parasite densities showed that high parasite densities increase the probability of detecting concurent clones in an individual [bib_ref] Typing of field isolates of Plasmodium falciparum, Contamin [/bib_ref]. Expectedly, a positive correlation between MOI and parasite density was found in the present study. The impact of sickle cell trait on MOI in this study is different from findings in Gabon by Ntoumi et al [bib_ref] Plasmodium falciparum: sickle-cell trait is associated with higher prevalence of multiple infections..., Ntoumi [/bib_ref] , in which, a correlation between sickle cell trait and MOI was found. However, this observation corroborates reports from Senegale by Konate et al [bib_ref] Mercereau-Puijalon O: Variation of Plasmodium falciparum msp1 block2 and msp2 allele prevalence..., Konate [/bib_ref] and Vafa et al [bib_ref] Migot-Nabias F: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal:..., Vafa [/bib_ref] whereby no correlation was found. These discrepancies can be explained by differences in age ranges of the study population and the number of individuals with sickle cell trait enrolled. The prevalence of pfcrt K76T was also assessed, the mutation associated with CQ resistance. It was anticipated that the sensitivity of the parasite to CQ may have been restored several years after its withdrawal as was the case in Malawi [bib_ref] Return of chloroquine antimalarial efficacy in Malawi, Laufer [/bib_ref]. The prevalence of Pfcrt K76T, an important molecular marker surveillance tool that can contribute to the evaluation of local parasite strains sensitivity to CQ in addition to in vitro studies [bib_ref] A requiem for chloroquine, Hasting [/bib_ref] , was 98% in Congo before withdrawal of CQ [bib_ref] In vivo chloroquine resistance and prevalence of the pfcrt codon 76 mutation..., Mayengue [/bib_ref]. Almost five years later, this study shows that the prevalence of this mutation remains high, 92%. This observation is similar to a recent report by Efunshile et al in Nigeria [bib_ref] Prevalence of molecular marker of chloroquine resistance (pfcrt 76) in Nigeria 5..., Efunshile [/bib_ref]. Therefore, CQ cannot be re-introduced in Congo now despite its many advantages over other anti-malarials. # Conclusion In summary, these findings shows that multiplicity of infection which is low in Congolese children with asymptomatic P. falciparum infection increases with parasite density. The prevalence of the mutant pfcrt allele remains extremely high in the study area. Further investigations with larger sample size in different zones of the country would be needed to confirm these observations. [fig] Figure 1: Distribution of P. falciparum 3D7 and FC27 msp2 alleles. [/fig] [fig] Figure 2: Correlation of msp2 multiplicity of infection and mean Plasmodium falciparum parasite density Spearman rank coefficient = 0.4489, P = 0.0278. [/fig] [table] Table 1: Demographic data and parasitological indexes of the study population [/table] [table] Table 2: Genetic diversity of P. falciparum msp2 gene [/table] [table] Table 3: Comparison of the parasite prevalence, parasite density and the MOI in different age groups and haemoglobin (Hb) phenotypes * Proportion of multiple infections in PCR positive samples, NS non significant [/table]

Effect of atorvastatin on iron metabolism regulation in patients with chronic kidney disease – a randomized double blind crossover study Introduction:To determine the effect of 6-month administration of atorvastatin on hepcidin and hemojuvelin levels, inflammatory parameters and iron metabolism in patients with chronic kidney disease (CKD) stages 3 and 4. Methods: Thirty six statin-and erythropoiesis-stimulating agent-naive patients with CKD stages 3 and 4 and LDL cholesterol !100 mg/dl received atorvastatin or placebo for two 6-month periods in a double blind, randomized crossover study. Hepcidin, hemojuvelin, hsCRP, IL-6, hemoglobin, red blood cell distribution width, iron, total iron binding capacity (TIBC), and unsaturated iron binding capacity (UIBC) were measured before and after each treatment period. Results: Hepcidin decreased (from 102 [307] to 63 [170] pg/ml (p > .001)) in the course of statin therapy but remained unchanged after placebo administration (173 [256] to 153 [204] pg/ml, respectively). Hemojuvelin did not change after either part of the study. Both IL-6 and hsCRP decreased following statin therapy (from 8.7 [12.0] to 8.1 [13.9] pg/ml; p ¼ .04 and from 4.7 [4.0] to 4.0 [3.6] mg/l; p ¼ .4, respectively), but did not change after placebo administration. Blood hemoglobin increased slightly but significantly after 6-month statin therapy (from 11.6 ± 1.6 to 11.9 ± 1.5 g/dl, p ¼ .002), and was unchanged after placebo treatment. TIBC and UIBC increased significantly after 6-month statin therapy, and serum iron also tended to increase. The change of eGFR during the study did not differ between the two treatment periods. Conclusions: Statin may have a small but potentially beneficial effect on serum hepcidin, which may lead to improvement of anemia control in CKD patients.ARTICLE HISTORY # Introduction Chronic kidney disease (CKD) is characterized by renal anemia caused by a relative deficiency of endogenous erythropoietin secretion, increased blood loss, and shortened red blood cell survival. CKD is also accompanied by mild to moderate chronic inflammation which may result in decreased availability of iron, and thereby contribute to the worsening of renal anemia [bib_ref] Defining a renal anemia management period, Besarab [/bib_ref]. Hepcidin is an endogenous antimicrobial peptide secreted by the liver [bib_ref] New insights into the regulation of iron homeostasis, Deicher [/bib_ref] [bib_ref] Hepcidin-a potential novel biomarker for iron status in chronic kidney disease, Zaritsky [/bib_ref]. Hepcidin serves as a critical determinant of iron metabolism, inhibiting intestinal absorption of dietary iron, release of iron from macrophages, and transfer of iron stored in hepatocytes. Hepcidin serves also as an acute phase protein and its expression is induced by inflammation [bib_ref] Hepcidin and iron regulation, 10 years later, Ganz [/bib_ref]. Serum hepcidin is increased in inflammatory states and in CKD, leading to decreased release of iron from macrophages resulting in insufficient erythropoiesis and anemia [bib_ref] Hepcidin: a new tool in the management of anaemia in patients with..., Swinkels [/bib_ref]. Animal studies have also shown that increased expression of hepcidin leads to a blunted erythropoietic response to endogenous epoetin [bib_ref] The role of hepcidin in iron metabolism, Nemeth [/bib_ref]. Hepcidin degrades ferroportin, which leads to accumulation of iron in macrophages and hepatocytes [bib_ref] The molecular mechanism of hepcidin-mediated ferroportin down-regulation, De Domenico [/bib_ref] [bib_ref] Hepcidin targets ferroportin for degradation in hepatocytes, Ramey [/bib_ref]. However, despite adequate deposits of iron, the available amount of circulating hemoglobin could be reduced. The hepcidin level is inversely correlated with the estimated glomerular filtration rate in patients with CKD, and its level is reduced during epoetin administration [bib_ref] Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal..., Ashby [/bib_ref]. Serum hepcidin concentration in patients with CKD increases progressively with the severity of CKD and can only be partly reduced by hemodialysis in endstage kidney disease [bib_ref] Is hepcidin a link between anemia, inflammation and liver function in hemodialyzed..., Małyszko [/bib_ref] [bib_ref] Prohepcidin accumulates in renal insufficiency, Taes [/bib_ref] [bib_ref] Urinary hepcidin: an inverse biomarker of acute kidney injury after cardiopulmonary bypass?, Prowle [/bib_ref] [bib_ref] Serum hepcidin in haemodialysis patients: associations with iron status and microinflammation, Xu [/bib_ref] [bib_ref] Impact of hepcidin, interleukin 6, and other inflammatory markers with respect to..., Ibrahim [/bib_ref]. It has been recently postulated that the increased production of hepcidin may be an important cause of resistance to erythropoietin stimulating agent (ESA) therapy in advanced or end-stage CKD [bib_ref] The role of hepcidin in iron metabolism, Nemeth [/bib_ref]. Hepcidin expression is regulated through the bone morphogenetic protein-hemojuvelin (BMP-HJV) signaling pathway. Hemojuvelin is a member of the repulsive guidance molecule family and appeared lately to be a key regulator of iron-dependent secretion of hepcidin. This iron-regulatory protein is mainly expressed in the liver, skeletal muscle, and heart and its production follows a specific process. The furin mRNA level is negatively regulated by iron concentrations [bib_ref] Soluble hemojuvelin is released by proprotein convertase-mediated cleavage at a conserved polybasic..., Lin [/bib_ref] [bib_ref] Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron..., Silvestri [/bib_ref]. The HJV produced by furin cleavage downregulates hepcidin expression by competing with hepatocyte membranebound mHJV for BMP binding. When not inhibited by HJV, this binding starts a cascade ending with an active protein complex that translocates into the nucleus and regulates hepcidin expression positively [bib_ref] The evaluation of hyperferritinemia: an updated strategy based on advances in detecting..., Aguilar-Martinez [/bib_ref] [bib_ref] Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and..., Lin [/bib_ref]. The inhibitors of 3-hydroxy-3-methy-glutaryl-co-enzyme A reductase reduce serum lipid levels and may have several other effects, including reduction of inflammation. Since statin therapy may overcome ESA hyporesponsiveness [bib_ref] Beneficial effect of atorvastatin on erythropoietin responsiveness in maintenance haemodialysis patients, Tsouchnikas [/bib_ref] , it might be postulated that the beneficial effect of statins on response to ESA therapy may be mediated by a decreased release of inflammatory mediators and increased release of iron from its stores [bib_ref] Beneficial effect of atorvastatin on erythropoietin responsiveness in maintenance haemodialysis patients, Tsouchnikas [/bib_ref] [bib_ref] Atorvastatin increases erythropoietin-stimulating agent hyporesponsiveness in maintenance hemodialysis patients: role of anti-inflammatory..., Chiang [/bib_ref]. This concept is supported by the results of the study which shows that administration of fluvastatin in patients with end-stage kidney disease leads to a decrease of prohepcidin, a prohormone of hepcidin [bib_ref] Effect of fluvastatin on serum prohepcidin levels in patients with endstage kidney..., Arabul [/bib_ref]. To the best of our knowledge, there have been no studies on the effect of statins on hepcidin and hemojuvelin levels, and thus iron metabolism and anemia in ESA naive patients with CKD not yet on dialysis. The aim of the study was to assess the effect of 6month administration of atorvastatin on hepcidin and hemojuvelin levels, inflammatory parameters and iron metabolism in patients with stages 3 and 4 CKD. # Materials and methods We obtained permission to perform this study from the Local Ethics Committee and followed the guidelines of the Helsinki Declaration. All the subjects were informed about the aims and design of the study, and provided a written informed consent prior to participation. The study was funded by the Medical University of Lodz research Grant No. 503/1-151-02/503-01. ## Patients and study design In a double-blind placebo-controlled crossover study, the patients randomly received either atorvastatin (20 mg in a single evening dose) or identically looking placebo tablets for the first 6 months. The treatment arms cross-changed after the wash-out period and the therapy with placebo or atorvastatin continued for another 6 months. The measurements were taken four times, i.e., prior to and at the end of each treatment period [fig_ref] Figure 1: Study design. [/fig_ref]. The patients were included in the study if they had a stable kidney function (±5%) for at least 6 months prior to the study and were not expected to start the renal replacement therapy within the next 6 months. All the patients were ESAs naïve, with adequate iron stores (ferritin level above >150 ng/ml and TSAT above 20%), with a constant dose of oral iron administered for at least the last 6 months, and not receiving any blood transfusion or intravenous iron for 3 months before the study. The doses of all other medications were unchanged throughout the study except for emergencies. Exclusion criteria included the hemoglobin level below 10 g/dl, uncontrolled or treatment-resistant hypertension [bib_ref] ESH/ESC guidelines for the management of arterial hypertension. The task force for..., Mancia [/bib_ref] , persistent urine protein excretion >1 g/24 h, or chronic steroid therapy with a dose of prednisone >5 mg/24 h, chronic heart failure NYHA stage 3 or higher, malignancy, administration of any lipid-lowering medication for 6 months prior to the study and administration of oral phosphate binders. Blood samples were obtained after overnight fasting, collected in EDTA tubes, and promptly centrifuged at 2000Âg at 2-8 C for 10 min within 30 min of sampling. The plasma was stored in aliquots at À70 C. The following biochemical parameters were measured at each visit: serum creatinine, total-, HDL-, LDL-cholesterol, triglyceride, uric acid, total protein, albumin, hepcidin, hemojuvelin, iron, ferritin, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), hemoglobin, red blood cell distribution width (RDW), C-reactive protein, as well as alanine and aspartate aminotransferase. Plasma hemojuvelin and hepcidin concentrations were measured using a commercial immunoassay from USCN Life Science (Houston, TX). All the other parameters were measured with standard laboratory automated methods in the local laboratory. TIBC values were calculated from serum iron and UIBC, both of which were measured with colorimetric methods. RDW-CV calculation was based on both the width of the distribution curve and the mean cell size. It was calculated by an automated hematology analyzer by dividing the standard deviation of the mean cell size by the MCV of the red cells and multiplying by 100 to convert to a percentage. The results are expressed as mean ± SD or median ± interquartile ratio (IQR) dependent on the normality of variable distribution. Statistical significance was defined as p<.05. The normality of data was checked by the Kolmogorov-Smirnov test. The paired ttest was used to analyze the differences between statin and placebo. In the case of non-normally distributed variables, the Wilcoxon test was used. Pearson's correlation coefficient was calculated to assess the associations between the variables. Statistical analysis was performed using Statistica for Windows (version 10PL, StatSoft, Tulsa, OK). # Results The participants were selected from the population treated in the outpatient clinic of the tertiary nephrology center. After prescreening, 60 potentially illegible patients were identified in our database. After screening, 36 patients were included and all of them completed the study visits according to the protocol. The study population comprised 19 women and 17 men, mean age 56 ± 11 years with CKD stages 3 and 4. The clinical characteristics of the study population are presented in [fig_ref] Table 1: Baseline clinical and biochemical characteristics of the study group [/fig_ref]. The causes of CKD included chronic glomerular disease in 12 patients, diabetic kidney disease in 8, adult polycystic kidney disease in 4, arterial hypertension in 4, tubulointerstitial nephritis in 1, and unknown causes in seven patients. Eight patients suffered from type 2 diabetes, arterial hypertension was diagnosed in 34 patients, and ischemic heart disease in five patients. No clinically important side-effects of statin therapy were observed with the exception of a transient mild muscle pain in one of the patients at the beginning of atorvastatin therapy during the study. During the entire study periods, no acute illnesses including infections of such severity that in the opinion of the investigators could affect the course of the study were observed at scheduled visits. [fig_ref] Table 1: Baseline clinical and biochemical characteristics of the study group [/fig_ref] shows the clinical and biochemical characteristics of the participants. The effects of atorvastatin and placebo on serum concentration of hepcidin, hemojuvelin, hemoglobin, and iron metabolism, i.e., serum iron, ferritin, TSAT, TIBC, UIBC, red cell distribution width (RDW), are shown in [fig_ref] Table 2: Hematological and inflammatory parameters and kidney function during the study [/fig_ref]. There was a small but significant increase of serum hemoglobin in the course of atorvastatin .03 0.7 ± 3.9 (95% Cl -0.6 to 2.0) .5 Results are expressed as mean ± SD in the case of non-normally distributed variables or [median (IQR)] in the case of non-normally distributed variables. eGFR: estimated glomerular filtration rates -calculated using the MDRD abbreviated formula; UIBC: unsaturated iron binding capacity; TSAT: transferrin saturation; TIBC: total iron binding capacity; IL-6: interleukin-6. administration (from 11.6 ± 1.6 g/dl to 11.9 ± 1.5 g/dl; p ¼ .002). Similar effects were also observed in the case of UIBC and TIBC. There was a significant decrease of serum hepcidin concentration at the end of statin therapy compared to the baseline. All these parameters did not change significantly during placebo administration. Serum ferritin, TSAT, RDW, and hemojuvelin concentration did not change in either part of the study. Kidney function (eGFR) significantly decreased in both study periods [fig_ref] Table 2: Hematological and inflammatory parameters and kidney function during the study [/fig_ref] , i.e., during administration of atorvastatin (from 22.7 ± 9.4 to 21.0 ± 7.9 ml/min/ 1.73 m 3 , p ¼ .01) and during administration of placebo .09 -2.9 ± 9.2 (95% CI -5.9 to 0.1) .2 Results are expressed as mean ± SD in the case of non-normally distributed variables or [median (IQR)] in the case of non-normally distributed variables. AspAT: aspartate aminotransferase; AlAT: alanine aminotransferase. (22.1 ± 8.9 to 21.1 ± 8.7, p ¼ .005). There was no significant difference between mean changes of eGFR between both periods of the study (p ¼ .3). [fig_ref] Table 2: Hematological and inflammatory parameters and kidney function during the study [/fig_ref] shows also the inflammation parameters assessed during the study. Both hsCRP and IL-6 decreased significantly in the course of treatment with atorvastatin, but remained unchanged during the placebo treatment period. [fig_ref] Figure 2: Changes of UIBC, TIBC, hepcidin, hemoglobin, MCHC, and IL-6 after placebo and... [/fig_ref] shows the changes of UIBC, TIBC, hepcidin, hemoglobin, MCHC, and IL-6 after placebo and atorvastatin treatment. Total cholesterol, LDL-cholesterol, triglyceride, and uric acid decreased significantly and HDL-cholesterol increased significantly after atorvastatin therapy [fig_ref] Table 3: Serum concentration of cholesterol, HDL, LDL, triglycerides, uric acid, as well as... [/fig_ref]. These parameters did not significantly change after placebo treatment. Serum concentration of alanine and aspartate aminotransferase increased significantly after atorvastatin but not after placebo therapy [fig_ref] Table 3: Serum concentration of cholesterol, HDL, LDL, triglycerides, uric acid, as well as... [/fig_ref]. No significant correlations were found between serum hepcidin, IL-6 and ferritin at baseline (r ¼ .37, p ¼ .9; r ¼ .058, p ¼ .7, respectively) and between the changes of serum hepcidin, IL-6 and ferritin after atorvastatin therapy (r ¼ -0.316, p ¼ .06; r ¼ .006, p ¼ .9, respectively) and after placebo treatment (r ¼ -0.32, p ¼ .06; r ¼ .01, p ¼ .9, respectively). There was a significant positive correlation between serum hepcidin and hsCRP (r ¼ 0.37; p ¼ .025) at baseline. The changes of serum hepcidin and changes of hsCRP during atorvastatin therapy (r ¼ .13, p ¼ .4) and placebo administration (r ¼ .022, p ¼ .2) did not correlate significantly. # Discussion A chronic inflammatory state may be an additional factor contributing to increased expression of acute phase proteins such as hepcidin and ferritin. There are two major patterns of acute-phase responses in hepatocytes [bib_ref] Acute-phase proteins and other systemic responses to inflammation, Gabay [/bib_ref] [bib_ref] Cytokines and the hepatic acute phase response, Moshage [/bib_ref]. Type 1 response is induced by IL-1-like cytokines, whereas type 2 response is induced by IL-6. Although some studies showed a significant correlation between serum hepcidin and ferritin in patients with different disorders affecting iron metabolism [bib_ref] The gene encoding the iron regulatory peptide hepcidin is regulated by anemia,..., Gael [/bib_ref] [bib_ref] Regulation of ferritin genes and protein, Torti [/bib_ref] , the pathways regulating their expression seem to be distinct. The conflicting findings may also result from different methods of measurement, since Ashby et al. [bib_ref] Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal..., Ashby [/bib_ref] used a radioimmunoassay, while we used an enzyme-linked immunoassay. It is of note that we revealed a significant positive correlation between hepcidin and CRP at baseline, while no relation was found in the case of IL-6. That finding was consistent with the results of van der Weerd et al. [bib_ref] Hepcidin-25 in chronic hemodialysis patients in related to residual kidney function and..., Van Der Weerd [/bib_ref] , though that study was conducted in patients who were already on chronic HD. Some other studies also reported significant positive correlations between serum hepcidin and CRP or IL-6 in CKD patients as well as in subjects with fully preserved renal function [bib_ref] Hepcidin-a potential novel biomarker for iron status in chronic kidney disease, Zaritsky [/bib_ref] [bib_ref] Hepcidin, a putative mediator of anemia of inflammation, is a type II..., Nemeth [/bib_ref] [bib_ref] Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its..., Nemeth [/bib_ref]. Zaritsky et al. [bib_ref] Hepcidin-a potential novel biomarker for iron status in chronic kidney disease, Zaritsky [/bib_ref] found a significant correlation between serum hsCRP and hepcidin at all stages of CKD. Their finding may provide the evidence for the existence of a pathophysiological connection between hepcidin and CRP. However, our findings are not concordant with the findings of Ashby et al. [bib_ref] Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal..., Ashby [/bib_ref] , who did not find any relation between serum hepcidin and inflammatory markers (hsCRP and IL-6) in both CKD and HD patients. However, this may be due to the use of a different assay to measure serum hepcidin. The relation between serum hepcidin and ferritin has been well established. Serum ferritin functions as a biomarker of both iron stores in the reticulo-endothelial system and as an acute phase protein. The same applies to serum hepcidin. Therefore, these two markers are expected to change in parallel. Interestingly, we did not observe any significant change of serum ferritin after statin treatment, while serum hepcidin decreased significantly. We also did not find any correlation between the changes of serum hepcidin and ferritin during statin therapy. Our findings contrast with the results of the study of Ashby et al. [bib_ref] Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal..., Ashby [/bib_ref] , who reported a significant correlation between serum hepcidin and ferritin in CKD patients not yet on dialysis but not in chronic hemodialysis patients. However, CKD patients in their study [bib_ref] Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal..., Ashby [/bib_ref] had a wide range of renal function impairment with estimated GFR 8-98 ml/min, while in our study CKD patients were at more advanced stages of kidney disease. Renal anemia is undoubtedly a multifactorial condition. Its treatment is multidirectional due to complex interactions between chronic inflammation, iron metabolism and relative or absolute erythropoietin deficiency. Our study was designed to explore the potential benefit of statin in the management of anemia in patients with CKD. We postulated that the effect of statin on blood hemoglobin might be mediated by its anti-inflammatory properties. The present study was cross-over randomized double-blind to allow a direct comparison of the effect of statin and placebo within the same group of patients. The effects of statin therapy on the inflammatory parameters observed in our patients are in agreement with several other studies carried out in CKD patients. The results of the seminal Study of Heart and Renal Protection (SHARP) encompassing 9270 patients with CKD (3023 on dialysis and 6247 not) showed that the lowering of LDL cholesterol with a combination of simvastatin and ezetimibe reduced the risk of major atherosclerotic events in CKD patients with different degrees of renal function impairment [bib_ref] The effect of lowering LDL cholesterol with simvastatin plus ezetimibe in patients..., Baigent [/bib_ref]. In other studies that focused on CKD stages 3 and 4, statins also proved their lipid-lowering and anti-inflammatory effect [bib_ref] Effects of lipid-lowering therapy with rosuvastatin on atherosclerotic burden in patients with..., Sawara [/bib_ref] [bib_ref] Effect of rosuvastatin on C-reactive protein and renal function in patients with..., Verma [/bib_ref] [bib_ref] Effects of atorvastatin on inflammatory and fibrinolytic parameters in patients with chronic..., Goicoechea [/bib_ref]. Panichi et al. [bib_ref] In vivo and in vitro effects of simvastatin on inflammatory markers in..., Panichi [/bib_ref] found that 6 months of simvastatin therapy with a daily dose of 40 mg in stages 3 and 4 CKD caused a significant decrease of serum inflammatory markers CRP and IL-6. Similarly, in the study of Goicoechea et al. [bib_ref] Effects of atorvastatin on inflammatory and fibrinolytic parameters in patients with chronic..., Goicoechea [/bib_ref] , 6 months of atorvastatin administration at the same dose of 20 mg/day as in our study led to a significant decrease of CRP, TNF-a, and IL-1b. In another study, the most potent HMG-CoA reductase inhibitor rosuvastatin induced a similar anti-inflammatory effect in CKD patients [bib_ref] Effect of rosuvastatin on C-reactive protein and renal function in patients with..., Verma [/bib_ref]. Our study showed only a tendency of serum inflammatory markers such as CRP and IL-6 to decrease after statin therapy, and this effect was not significant in comparison with changes of these parameters after placebo therapy. Although HMG-CoA reductase inhibitors are considered generally safe in early CKD, there are differences in their pharmacokinetic properties that might determine the choice of a particular statin in advanced CKD or in end-stage renal disease. We have chosen atorvastatin for our study because it has negligible (<2%) renal eliminationand does not require any dose adjustment even in patients with eGFR below 30 ml/min/ 1.73 m 2 . We have selected a mid-range dose of atorvastatin since our patients had only moderate serum lipid abnormalities and the same dose was used in earlier studies in CKD [bib_ref] Effects of atorvastatin on inflammatory and fibrinolytic parameters in patients with chronic..., Goicoechea [/bib_ref]. Both after statin and placebo therapy, we observed a deterioration of eGFR, there was, however, no significant difference between mean changes of eGFR in both periods of the study. Our findings corroborate the results of previous studies including the largest SHARP trial [bib_ref] The effect of lowering LDL cholesterol with simvastatin plus ezetimibe in patients..., Baigent [/bib_ref] that showed no effect of statin on kidney disease progression. The effect may be however dependent on a dose of statin. One recent metaanalysis [bib_ref] Effects of statins on renal outcome in chronic kidney disease patients: a..., Sanguankeo [/bib_ref] showed that high-intensity statin therapy slightly slowed a decline in eGFR in population with CKD compared with control, but moderate-and low-intensity statins, i.e., 10-20 mg/24 h atorvastatin, did not. We used moderate-intensity statin and that therapy was not found to have any significant effect on eGFR compared to placebo. In order to exclude the effect of ESA on iron metabolism, all the patients in our study were ESA naive with the baseline hemoglobin concentration over 10 g/dl, with adequate iron stores, and with a constant dose of oral iron therapy for at least the last 6 months. We therefore postulate that the increase of hemoglobin during administration of atorvastatin with a significant increase of TIBC and UIBC may be most likely explained in our study by the anti-inflammatory action of statin leading to decreased expression of hepcidin. The fact that a change of eGFR, which is a major determinant of renal anemia, was similar after atorvastatin and placebo in our patients may confirm the concept. The RDW reflects the variability in size of circulating red blood cells and is routinely reported by automated blood counts in laboratories, though it is rarely interpreted by physicians. Most studies have shown that RDW increases in the presence of systemic inflammation [bib_ref] RDW in patients with systemic lupus erythematosus. Influence of anemia and inflammatory..., Vaya [/bib_ref] [bib_ref] Red blood cell distribution width is a potential index to assess the..., Hu [/bib_ref] [bib_ref] Association between red cell distribution width and disease activity in patients with..., Song [/bib_ref]. Moreover, RDW has been strongly associated with increased serum inflammatory markers and disease activity in patients with systemic lupus erythematosus [bib_ref] RDW in patients with systemic lupus erythematosus. Influence of anemia and inflammatory..., Vaya [/bib_ref] [bib_ref] Association between red cell distribution width and disease activity in patients with..., Song [/bib_ref] and with inflammatory bowel disease [bib_ref] Association between red cell distribution width and disease activity in patients with..., Song [/bib_ref] [bib_ref] Red cell distribution width for assessment of activity of inflammatory bowel disease, Cakal [/bib_ref]. In a study involving almost 5000 outpatients [bib_ref] Association of red blood cell distribution width with plasma lipids in a..., Lippi [/bib_ref] , a significant association of increased RDW with low HDL-C and high levels of TG was reported. In our study, we did not find any significant changes of RDW after statin treatment, despite a consistent lipid-lowering effect. These results are consistent with the study of Kucera et al. [bib_ref] Effect of atorvastatin on low-density lipoprotein subpopulations and comparison between indicators of..., Kucera [/bib_ref] , in which a higher dose of atorvastatin 40 mg/24 h was used and the time of the treatment was two times shorter than in our study. Akin et al. [bib_ref] Effect of atorvastatin on hematologic parameters in patients with hypercholesterolemia, Akin [/bib_ref] also did not find any effect of 10-80 mg per day of atorvastatin on RDW. Additionally, we observed a significant decrease of serum uric acid after statin. This observation is consistent with the study of Milionis et al. [bib_ref] Effects of statin treatment on uric acid homeostasis in patients with primary..., Milionis [/bib_ref] , who studied the effect of atorvastatin and simvastatin on uric acid homeostasis in patients treated for primary hyperlipidemia. Interestingly, in that study only atorvastatin, but not simvastatin, had a significant effect on serum uric acid [bib_ref] Effects of statin treatment on uric acid homeostasis in patients with primary..., Milionis [/bib_ref]. The main limitation of our exploratory research was a relatively small group of patients and a possible interference of the progression of CKD on the parameters of anemia and iron metabolism during the study; however, the cross-over design of the study and randomization allowed intra-individual comparison of the effects of an active therapy and placebo. Due to the design of the study that including a long washout period that exceeded several times T1/2 of the drug the risk of carry-over effect has been minimized. Since the subject clinical characteristics did not change during the whole duration of the study and none of the patients suffered from new or worsening diseases or underwent acute clinically relevant infections during the study, the interference of the period effect on the results was also eliminated. Additionally, we did not investigate the effect of atorvastatin on blood pressure and proteinuria, and we did not compare effects of different statins. To the best of our knowledge, the present study is the first to investigate the effect of statin therapy on hepcidin and hemojuvelin serum concentration, iron utilization and metabolism in ESA naive patients with moderate to advanced CKD. # Conclusions Treatment with statin may have potentially beneficial effects on the hepcidin level, and thus on iron metabolism in this population. Although the lipid-lowering effect of atorvastatin was only moderate, it may also have a potentially beneficial effect on hepcidin serum concentration, and thus on the inflammatory state, iron utilization and anemia in patients with CKD. # Disclosure statement No potential conflict of interest was reported by the authors. [fig] Figure 1: Study design. [/fig] [fig] Figure 2: Changes of UIBC, TIBC, hepcidin, hemoglobin, MCHC, and IL-6 after placebo and atorvastatin treatment. [/fig] [table] Table 1: Baseline clinical and biochemical characteristics of the study group.Results are expressed as mean ± SD [median (IQR)]. a eGFR: estimated glomerular filtration ratescalculated using the MDRD abbreviated formula; UIBC: unsaturated iron binding capacity; TSAT: transferrin saturation; TIBC: total iron binding capacity; CRP: C-reactive protein; AspAT: aspartate aminotransferases; AlAT: alanine aminotransferases; IL-6: interleukin-6. [/table] [table] Table 2: Hematological and inflammatory parameters and kidney function during the study. [/table] [table] Table 3: Serum concentration of cholesterol, HDL, LDL, triglycerides, uric acid, as well as aspartate and alanine aminotransferase during the study. [/table]

Elevated Striatal Dopamine Function in Immigrants and Their Children: A Risk Mechanism for Psychosis Migration is a major risk factor for schizophrenia but the neurochemical processes involved are unknown. One candidate mechanism is through elevations in striatal dopamine synthesis and release. The objective of this research was to determine whether striatal dopamine function is elevated in immigrants compared to nonimmigrants and the relationship with psychosis. Two complementary case-control studies of in vivo dopamine function (stress-induced dopamine release and dopamine synthesis capacity) in immigrants compared to nonimmigrants were performed in Canada and the United Kingdom. The Canadian dopamine release study included 25 immigrant and 31 nonmigrant Canadians. These groups included 23 clinical high risk (CHR) subjects, 9 antipsychotic naïve patients with schizophrenia, and 24 healthy volunteers. The UK dopamine synthesis study included 32 immigrants and 44 nonimmigrant British. These groups included 50 CHR subjects and 26 healthy volunteers. Both striatal stress-induced dopamine release and dopamine synthesis capacity were significantly elevated in immigrants compared to nonimmigrants, independent of clinical status. These data provide the first evidence that the effect of migration on the risk of developing psychosis may be mediated by an elevation in brain dopamine function. # Introduction Geographic mobility has a large impact on sociological, economic, and health factors within communities. Over 247 million people, or 3.5% of the world population, became migrants over the last decade.One of the most consistent findings in the epidemiology of schizophrenia is the high incidence of the disorder among immigrant groups (relative risk vs nonimmigrant: 2.9). [bib_ref] Schizophrenia and migration: a meta-analysis and review, Cantor-Graae [/bib_ref] The risk is particularly increased in immigrant groups who migrate from a country where the population is predominantly black skinned to a country where the population is predominantly white skinned (relative risk vs nonimmigrant: 4.8). [bib_ref] Schizophrenia and migration: a meta-analysis and review, Cantor-Graae [/bib_ref] This increased risk of schizophrenia has been reported both in immigrants and in their children. These findings have been replicated in a number of high income countries: The Netherlands, [bib_ref] Discrimination and the incidence of psychotic disorders among ethnic minorities in The..., Veling [/bib_ref] Denmark, [bib_ref] Risk of schizophrenia in second-generation immigrants: a Danish population-based cohort study, Cantor-Graae [/bib_ref] Germany, [bib_ref] Schizophrenic disorders among Turkish migrants in Germany. A controlled clinical study, Haasen [/bib_ref] the United Kingdom, [bib_ref] Increased incidence of psychotic disorders in migrants from the Caribbean to the..., Harrison [/bib_ref] and Canada, [bib_ref] Incidence of psychotic disorders among first-generation immigrants and refugees in Ontario, Anderson [/bib_ref] clearly establishing that the incidence of schizophrenia is higher among migrant groups as compared to host populations. Models of how social factors such as immigration lead to psychosis have mainly considered the role of stress. [bib_ref] Schizophrenia: an integrated sociodevelopmental-cognitive model, Howes [/bib_ref] Dysregulation of the stress response is a potential etiological factor in the development and relapse of dopamine (DA)-related human disorders including drug-induced psychosis and schizophrenia. [bib_ref] PET studies of binding competition between endogenous dopamine and the D1 radiotracer..., Abi-Dargham [/bib_ref] [bib_ref] Behavioural sensitization to daily life stress in psychosis, Myin-Germeys [/bib_ref] The stress-vulnerability model suggests that an endogenous, organic diathesis or This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. vulnerability interacts with internal or external stressors in the development of psychotic disorders. [bib_ref] Vulnerability-a new view of schizophrenia, Zubin [/bib_ref] This includes the social defeat hypothesis, which suggests that social defeat stress may lead to psychosis through sensitization of striatal DA neurotransmission. [bib_ref] Social defeat: risk factor for schizophrenia?, Selten [/bib_ref] [bib_ref] The social defeat hypothesis of schizophrenia: an update, Selten [/bib_ref] Initial studies in healthy volunteers have examined the impact of various forms of social defeat on brain function, [bib_ref] City living and urban upbringing affect neural social stress processing in humans, Lederbogen [/bib_ref] [bib_ref] Neuroimaging evidence for a role of neural social stress processing in ethnic..., Akdeniz [/bib_ref] [bib_ref] Increased release of dopamine in the striata of young adults with hearing..., Gevonden [/bib_ref] [bib_ref] History of childhood adversity is positively associated with ventral striatal dopamine responses..., Oswald [/bib_ref] including striatal DA release. [bib_ref] Increased release of dopamine in the striata of young adults with hearing..., Gevonden [/bib_ref] [bib_ref] History of childhood adversity is positively associated with ventral striatal dopamine responses..., Oswald [/bib_ref] Two recent studies showed that urban living [bib_ref] City living and urban upbringing affect neural social stress processing in humans, Lederbogen [/bib_ref] and immigration [bib_ref] Neuroimaging evidence for a role of neural social stress processing in ethnic..., Akdeniz [/bib_ref] were associated with altered brain responses to stress, providing the first link between social factors and brain function. Particularly in immigrants, perceived discrimination of participant's ethnic group predicted activation of the ventral striatum, [bib_ref] Neuroimaging evidence for a role of neural social stress processing in ethnic..., Akdeniz [/bib_ref] an area with dense DA innervation. However, so far no study has directly investigated dopamine function in immigrants. DA hyperactivity in the striatum is a core neurobiological feature of schizophrenia that could underlie the exaggerated incidence of schizophrenia in immigrants. [bib_ref] Presynaptic striatal dopamine dysfunction in people at ultrahigh risk for psychosis: findings..., Egerton [/bib_ref] [bib_ref] Progressive increase in striatal dopamine synthesis capacity as patients develop psychosis: a..., Howes [/bib_ref] [bib_ref] Elevated striatal dopamine function linked to prodromal signs of schizophrenia, Howes [/bib_ref] [bib_ref] Increased synaptic dopamine function in associative regions of the striatum in schizophrenia, Kegeles [/bib_ref] [bib_ref] Increased stressinduced dopamine release in psychosis, Mizrahi [/bib_ref] Here, we tested this hypothesis directly using positron emission tomography (PET) in vivo in immigrants and their children with, or at risk for, schizophrenia. To further validate our finding we report two complementary PET DA neuroimaging approaches to study two immigrant populations, one in Canada and one in the United Kingdom. The Canadian study investigated whether the induction of stress by a validated laboratory psychosocial task (Montreal Imaging Stress Task, MIST) 24 elicits more DA release in immigrants (first and second generation) as compared to the host population. The UK study sought to confirm the association between immigration (first and second generation) and elevated striatal DA function in a different cohort using the complementary technique of [ 18 F]DOPA PET to estimate DA synthesis capacity. 25 # Methods ## Canada study: stress-induced striatal dopamine release Participants. All subjects provided written, informed consent to participate. Immigration status determined using the self-reported place of birth of the participants, their parents, and grandparents. The immigrant group (N = 26) included first (N = 9) and second-generation immigrants (N = 8) to Canada (The generation information details were not available in 9 subjects, supplementary table S1). The nonimmigrant group (N = 31) had been in Canada for at least 3 generations. The immigrant and nonimmigrant groups were comparable for demographics [fig_ref] Table 1: Demographic Data by Immigration Status for the Stress-Induced DA Release Study, Canada... [/fig_ref]. The immigrant and nonimmigrant groups included subjects at clinical high risk (CHR) for psychosis (N = 23), antipsychotic naïve patients with schizophrenia (SCZ, N = 9) and healthy volunteers (HV, N = 24) (supplementary table S1). The CHR subjects met criteria for prodromal syndromes [bib_ref] Prodromal assessment with the structured interview for prodromal syndromes and the scale..., Miller [/bib_ref] based on the Structured Interview of Prodromal Syndromes (SIPS) [bib_ref] Prodromal assessment with the structured interview for prodromal syndromes and the scale..., Miller [/bib_ref] [bib_ref] Prospective diagnosis of the initial prodrome for schizophrenia based on the Structured..., Miller [/bib_ref] and were recruited from the early identification and treatment services for those who are at risk of psychosis (Focus on Youth Psychosis Prevention clinic, FYPP) at the Centre for Addiction and Mental Health (CAMH), Toronto, Canada. SCZ subjects were recruited from the first episode psychosis clinic at CAMH, with the diagnosis of schizophrenia and schizophreniform disorder, no other current Axis I psychotic disorders, and no antipsychotic exposure (ie, antipsychotic naïve). HV were recruited from the same geographic area, had no personal history of psychiatric symptoms, and were not taking psychotropic medication. Participants from the current study overlapped with the cohort in previous dopamine imaging studies [bib_ref] Increased stressinduced dopamine release in psychosis, Mizrahi [/bib_ref] [bib_ref] Stress-induced dopamine response in subjects at clinical high risk for schizophrenia with..., Mizrahi [/bib_ref] [bib_ref] Dopamine D2 and D3 binding in people at clinical high risk for..., Suridjan [/bib_ref] in whom information on immigration status was available. Exclusion criteria for all participants included pregnancy, other contra-indications to PET imaging and illicit drug use other than cannabis in the 6 months prior to imaging. The absence of illicit substance use other than cannabis was confirmed using urine drug screens at the time of PET imaging. In all participants, the level of perceived stress was measured using the Trier Inventory for the Assessment of Chronic Stress. [bib_ref] Trierer Inventar zur Erfassung von chronischem Streß (TICS): Skalenkonstruktion, teststatistische Überprüfung und..., Schulz [/bib_ref] Montreal Imaging Stress Task. A psychological stress task was performed during the PET imaging session to elicit dopamine release. Psychological stress was induced using the Montreal Imaging Stress Task (MIST) which has been validated in previous fMRI and PET studies [bib_ref] City living and urban upbringing affect neural social stress processing in humans, Lederbogen [/bib_ref] [bib_ref] Neuroimaging evidence for a role of neural social stress processing in ethnic..., Akdeniz [/bib_ref] [bib_ref] Increased stressinduced dopamine release in psychosis, Mizrahi [/bib_ref] [bib_ref] Dopamine release in response to a psychological stress in humans and its..., Pruessner [/bib_ref] [bib_ref] Stress-induced dopamine response in subjects at clinical high risk for schizophrenia with..., Mizrahi [/bib_ref] [bib_ref] The Montreal Imaging Stress Task: using functional imaging to investigate the effects..., Dedovic [/bib_ref] [bib_ref] Deactivation of the limbic system during acute psychosocial stress: evidence from positron..., Pruessner [/bib_ref] (see supplementary e-material MIST section for details). In all experiments, the control or stress task was started ~6-8 min before tracer injection, with 6 min of mathematical questions and ~1-2 min for either neutral or negative verbal feedback. Perception of stress during the control and stress conditions was assessed by a shortened version of the state anxiety questionnaire. [bib_ref] Increased stressinduced dopamine release in psychosis, Mizrahi [/bib_ref] [bib_ref] Stress-induced dopamine response in subjects at clinical high risk for schizophrenia with..., Mizrahi [/bib_ref] The stress task was effective in producing subject-tailored failure and eliciting a stress response, which was equivalent across the immigrant and nonimmigrant groups (supplementary figures S1 and S2). [ 11 C]-(+)-PHNO PET Image Acquisition. All 56 subjects completed 2 PET scans (n = 112 PET scans) at least a week apart at the same time of the day, one while performing the SMCT (sensory motor control task) and one while undergoing the MIST (stress task). Stress-induced DA release was measured using [ 11 C]-(+)-PHNO positron emission tomography (PET), through quantification of the competition between endogenous DA and [ 11 C]-(+)-PHNO for D2/3 receptor binding in the striatum. The radiosynthesis of [ 11 C]-(+)-PHNO has been described in detail elsewhere. [bib_ref] Radiosynthesis and evaluation of [11C]-(+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol as a potential radiotracer for in vivo..., Wilson [/bib_ref] Studies were carried out using a high resolution PET CT, Siemens-Biograph HiRez XVI (Siemens Molecular Imaging ). Each subject was administered ~333-370 MBq of high specific activity [ 11 C]-(+)-PHNO and scanned for 90 min. A custom-fitted thermoplastic mask was made for each subject and used with a head fixation system during PET acquisition to minimize head movement. A CT transmission scan was acquired after each emission scan for attenuation correction. [ 11 C]-(+)-PHNO PET Image Analysis. PET images were reconstructed with a 2D filtered back projection algorithm with a ramp filter at Nyquist cut-off frequency and rebinned into 31 time frames (comprising the background frame, followed by [ 11 C]-(+)-PHNO injection by fifteen 60-s frames and fifteen 300-s frames) as previously validated.Time activity curves (TAC) from the regions of interest (ROIs) were obtained from the dynamic [ 11 C]-(+)-PHNO PET images. The ROIs selected were the whole striatum and its functional subdivisions, including the associative (AST), limbic (LST), and sensorimotor striatum (SMST). [bib_ref] Imaging human mesolimbic dopamine transmission with positron emission tomography. Part II: amphetamine-induced..., Martinez [/bib_ref] ROIs were delineated using an automated method implemented in an in-house software (ROMI). [bib_ref] An automated method for the extraction of regional data from PET images, Rusjan [/bib_ref] Activity from bilateral ROI were combined and the volume-weighted average signal was used to derive [ 11 C]-(+)-PHNO binding potential (BP ND ). [bib_ref] Consensus nomenclature for in vivo imaging of reversibly binding radioligands, Innis [/bib_ref] BP ND was calculated by the Simplified Reference Tissue Method 39 using a cerebellar reference region, as previously described for [ 11 C]-(+)-PHNO. [bib_ref] Positron emission tomography quantification of [11C]-(+)-PHNO binding in the human brain, Ginovart [/bib_ref] Stress-induced DA release was quantified as [formula] [ 11 C]-(+)- PHNO % displacement = (BP ND SMCT − BP ND MIST) / BP ND SMCT) × 100%. [/formula] In complement to the ROI approach, stress-induced DA release was also assessed using voxel-wise analysis. Each BP ND parametric map was spatially normalized to the Montreal Neurological Institute (MNI) anatomical template using SPM2 normalization and co-registration tools. These maps were then used to assess significant contrasts between nonstress and stress conditions at the level of the whole brain using an implicit mask of BP ND >0.3. This mask restricts the statistical search to areas of specific binding (ie, excluding cerebrospinal fluid, background, and the reference region). ## Uk study: striatal dopamine synthesis capacity Participants. All subjects provided written, informed consent to participate. As in the Canada study, immigration status determined using the self-reported place of birth of the participants, their parents, and grandparents. The immigrant group (N = 31) included first (N = 13) and second-generation (N = 18) immigrants to the United Kingdom. The nonimmigrant group (N = 44) had been in the United Kingdom for at least 3 generations. The immigrant and nonimmigrant groups were comparable for demographics [fig_ref] Table 2: Demographic Data by Immigration Status for the DA Synthesis Study, UK Site [/fig_ref]. These participants included 50 individuals who met operationalized CHR criteria 41 and 26 HV [fig_ref] Table 2: Demographic Data by Immigration Status for the DA Synthesis Study, UK Site [/fig_ref]. CHR participants were recruited from Outreach and Support in South London (OASIS, part of the South London and Maudsley National Health Service Trust). Of the 50 CHR subjects, 2 were using antipsychotics (1 using olanzapine and 1 using quetiapine). The HV group was recruited from the same geographic area and had no personal of psychiatric symptoms, or psychotropic medication. Both the CHR and HV groups included subjects who had participated in previous dopamine imaging studies [bib_ref] Presynaptic striatal dopamine dysfunction in people at ultrahigh risk for psychosis: findings..., Egerton [/bib_ref] [bib_ref] Elevated striatal dopamine function linked to prodromal signs of schizophrenia, Howes [/bib_ref] where information on immigration status was available. Exclusion criteria for all participants included pregnancy or other contra-indications to PET imaging. The absence of illicit substance use other than cannabis was confirmed by urine drugs screen at the time of PET imaging. [ 18 F]-DOPA PET Image Acquisition. Studies were carried out using either a CTI/Siemens ECAT HR+ 962 tomograph (16 HV; 26 CHR) [bib_ref] Presynaptic striatal dopamine dysfunction in people at ultrahigh risk for psychosis: findings..., Egerton [/bib_ref] or a CTI/Siemens ECAT HR++ 966 tomograph (10 HV; 24 CHR) [bib_ref] Elevated striatal dopamine function linked to prodromal signs of schizophrenia, Howes [/bib_ref] (Siemens Molecular Imaging). All participants were asked to fast for 12 h before imaging and received carbidopa (150 mg) and entacapone (400 mg) orally 1 h before imaging to [ 18 F]-DOPA PET Image Analysis. Data were reconstructed using the 3D reprojection algorithms. To correct for head movement during the scan, nonattenuation-corrected dynamic images were denoised using a level 2, order 64 Battle-Lemarie wavelet filter [bib_ref] Multiresolution analysis of emission tomography images in the wavelet domain, Turkheimer [/bib_ref] and individual frames were realigned using a mutual information algorithm. [bib_ref] Automated 3-D registration of MR and CT images of the head, Studholme [/bib_ref] The transformation parameters were then applied to the corresponding attenuation-corrected frames, and the realigned frames were combined to create a movementcorrected dynamic image for analysis. Striatal ROI were delineated bilaterally on a single subject T1 MRI in MNI space. The cerebellar reference region was defined using a probabilistic atlas. [bib_ref] Three-dimensional maximum probability atlas of the human brain, with particular reference to..., Hammers [/bib_ref] An [ 18 F]-DOPA template, also in MNI space, was then normalized together with the ROI map to each individual PET summation image using SPM5. [bib_ref] The test-retest reliability of 18F-DOPA PET in assessing striatal and extrastriatal presynaptic..., Egerton [/bib_ref] Graphical analysis, adapted for a reference tissue input function [bib_ref] Graphical evaluation of blood-tobrain transfer constants from multiple-time uptake data, Patlak [/bib_ref] [bib_ref] Multiresolution Bayesian regression in PET dynamic studies using wavelets, Turkheimer [/bib_ref] was used to estimate presynaptic dopamine synthesis capacity by calculating the rate of utilization of the dopamine precursor 18 F-DOPA (k i cer min −1 ) in the bilateral striatum. To control for potential effects of scanner model, [bib_ref] Presynaptic striatal dopamine dysfunction in people at ultrahigh risk for psychosis: findings..., Egerton [/bib_ref] individual subject k i cer values were converted to z-scores [z = (k i cer -scanner mean k i cer ) / scanner standard deviation (SD)] for all analyses. # Statistical analysis Analysis of covariance was used to determine the main effects of immigration status and of clinical group on [ 11 C]-(+)-PHNO displacement or 18 F-DOPA z-score in the striatum. Nonsignificant immigration × clinical group interaction terms were subsequently removed from the model. Effect sizes were calculated as partial eta 2 . Follow-up analysis co-varied for potential effects of illicit drug use. The voxel-wise analysis of DA release utilized SPM8 to examine the difference between the control and stress conditions using paired t-tests in the immigrant and nonimmigrant groups, with cannabis use and clinical status as covariates. Partial correlation examined the relationship between perceived stress and [ 11 C]-(+)-PHNO displacement, co-varying for clinical status (n = 54). Statistical significance was defined as P < .05 unless otherwise stated. Secondary exploratory ANOVA comparing nonimmigrants to first and second generation immigrants separately found an overall significant effect of immigration on striatal DA release (F = 3.31; df = 2, 44; P = .046), with post hoc tests indicating this was primarily driven by elevated striatal DA release in the first generation immigrants compared to the nonimmigrant group (Bonferroni corrected comparisons, P = .05), whereas stress induced DA release did not differ significantly between second generation immigrants and nonimmigrants (P = .68) or first generation immigrant groups (P = 1.00). When the ROI analysis was repeated with cannabis use included as a covariate, the effects of both immigration (F = 7.26; df = 1, 51; P = .01) and clinical group (F = 3.68; df = 2, 51; P = .03) remained significant across the whole striatum. The immigration effect remained significant in AST (F = 7.23; df = 1, 51; P = .01) and LST (F = 4.48; df = 1, 51; P = .04), and trend level in SMST (F = 3.51; df = 1, 51; P = .07). The clinical group effect was also significant in AST (F = 5.15; df = 1, 51; P = .01), and was present at trend-level in SMST (F = 2.81; df = 2, 51; P = .07), but not in LST. The effects of immigration and clinical group on DA release also remained after co-varying for lifetime history of previous use of other illicit drugs [fig_ref] Table 1: Demographic Data by Immigration Status for the Stress-Induced DA Release Study, Canada... [/fig_ref]. Stress-induced striatal DA release was associated with several measures of social stress, including work overload, social overload, social pressure, social isolation, etc. (supplementary table S1 and supplementary . # Results ## Canada study: stress-induced striatal dopamine release ## Uk study: striatal dopamine synthesis capacity Striatal DA synthesis capacity was elevated in the immigrant compared to the nonimmigrant group (F = 4.95; df = 1, 73; P = .03, partial eta2 = 0.06,. The clinical group effect showed a trend for increased DA synthesis in the CHR compared to HV group (F = 3.79; df = 1, 73; P = .06; partial eta2 = 0.05). The clinical group by immigration status interaction term was nonsignificant. The significant elevation in DA synthesis capacity in immigrants was present in the SMST (F = 9.24; df = 1, 73; P = .003; partial eta2 = 0.11), with a similar trend in the AST (F = 3.28; df = 1, 73; P = .07; partial eta2 = 0.04). Secondary exploratory ANOVA comparing nonimmigrants to first and second generation immigrants separately found an overall significant effect of immigration on striatal dopamine synthesis capacity (F = 3.12; df = 1, 73; P = .047), with post hoc tests indicating this was primarily driven by elevated DA synthesis capacity in the second generation immigrant compared to nonimmigrant group (P = .04), whereas DA synthesis capacity did not differ significantly between first generation immigrant and nonimmigrant (P = .91) or second generation immigrant groups (P = .23). When cannabis use was included in the model, the main effects of both immigration and clinical status on DA synthesis capacity were significant for the whole striatum (F = 4.83; df = 1, 72; P = .03 and F = 4.35; df = 1, 72; P = .04, respectively). The immigration effect remained significant in SMST (F = 9.06; df = 1, 72; P = .004) and trend level in AST (F = 3.18; df = 1, 72; P = .07). The clinical group effect was significant in AST (F = 4.56; df = 1, 72; P = .04). The main effects of immigration and clinical group on DA synthesis also remained after co-varying for previous use of other illicit substances (table 2), or after excluding the 2 CHR subjects who were currently taking antipsychotics. # Discussion These results indicate that striatal DA function is elevated in both immigrants and their children, including those at risk for psychosis or with schizophrenia, confirming its relevance for psychotic disorders. The elevation in DA in immigrants was present with relatively large effect size in both the Canada and UK studies, which were performed in independent samples using 2 complementary approaches to imaging presynaptic DA function. As in previous studies of schizophrenia, 21 elevated DA in immigrants were most evident in dorsal striatal regions (AST and SMST). This suggests that adverse psychological, social, and environmental experiences associated with immigration may increase the risk of schizophrenia by influencing brain dopamine function, a key pathophysiological component of psychosis. The increased DA function in immigrants and their children did not appear to be influenced by cannabis exposure. [bib_ref] Stress-induced dopamine response in subjects at clinical high risk for schizophrenia with..., Mizrahi [/bib_ref] [bib_ref] Dopaminergic function in cannabis users and its relationship to cannabis-induced psychotic symptoms, Bloomfield [/bib_ref] In both experiments, we confirmed the absence of illicit drugs other than cannabis at the time of PET imaging by urine drugs screen. There were no significant differences in self-reported drug use and immigration effects remained significant when previous illicit drug use was included in the analyses. While lack of detailed illicit drug histories or hair analyses is a limitation, from the available data we believe that our findings were unlikely to be related to drug use. As both the UK and Canadian cohorts included participants of multiple ethnic groups and different generation (1st vs 2nd) distribution, we were unable to determine the effects of immigration accounting for ethnicity or generation status. Future studies could focus on the impact of ethnicity and immigration generation on dopamine function. While the reasons for the increased risk of schizophrenia and other psychoses in migrant groups are still unclear, there is increasing evidence that victimization, discrimination, social isolation, social defeat, and growing up in an urban environment may contribute. [bib_ref] Increased release of dopamine in the striata of young adults with hearing..., Gevonden [/bib_ref] [bib_ref] Understanding the excess of psychosis among the African-Caribbean population in England. Review..., Sharpley [/bib_ref] [bib_ref] Perceptions of disadvantage, ethnicity and psychosis, Cooper [/bib_ref] [bib_ref] Social experience and psychosis insights from studies of migrant and ethnic minority..., Morgan [/bib_ref] Our subjects came from 2 large cities (London, UK and Toronto, Canada) with relatively large immigrant populations. One avenue for future research is the extent by which social support interventions can mitigate the relationship between risk factors such as immigration, neurobiological markers, and poor mental health. Given the significant waves of immigration happening at this time both in Europe and the Americas, these interventions may become key as pre-emptive strategies. Cross-sectional studies such as ours cannot provide causal information about the associations between immigration, elevated dopamine function, and schizophrenia. However, studies in experimental animals have shown that experiences of social defeat stress can lead to striatal DA elevation. [bib_ref] Social defeat stress selectively alters mesocorticolimbic dopamine release: an in vivo microdialysis..., Tidey [/bib_ref] More broadly, our data are consistent with hypotheses linking social defeat to elevated DA and schizophrenia, [bib_ref] Social defeat: risk factor for schizophrenia?, Selten [/bib_ref] [bib_ref] The social defeat hypothesis of schizophrenia: an update, Selten [/bib_ref] [bib_ref] A new look at the neural diathesis-stress model of schizophrenia: the primacy..., Jones [/bib_ref] [bib_ref] Stress and the hypothalamic pituitary adrenal axis in the developmental course of..., Walker [/bib_ref] and reports of striatal DA elevations in adults with hearing impairment, 17 history of childhood abuse 18 or low parental care, [bib_ref] Dopamine release in response to a psychological stress in humans and its..., Pruessner [/bib_ref] which may all be forms of social defeat/stress. Our study further supports the relevance of the interaction between adverse social factors and striatal DA for psychotic disorders. # Conclusions The data from the present study identify a plausible biological mechanism that links the effects of migrant status to the risk of developing psychosis through elevated brain dopaminergic function. These findings suggest that interventions designed to reduce the psychosocial impact of being a migrant might be useful as a means of reducing the risk of psychotic illness. # Supplementary material Supplementary data are available at Schizophrenia Bulletin online. # Funding The Canadian study was funded by the Canadian Institute of Health Research (CIHR) and the National Association for Research on Schizophrenia and Depression (NARSAD). The UK study was funded by the Medical Research Council (MRC), grant number G0700995, and presents independent research supported by the National Institute of Health Research (NIHR), Biomedical Research Centre at South London, Maudsley NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the department of Health. Prevention (FYPP) clinic in Toronto and the members of the Outreach and Support in South London (OASIS) who were involved in the recruitment and clinical management of the CHR participants. We are also grateful to the PET team at the Research Imaging Center at CAMH, in Toronto, Canada and at the Cyclotron Unit, Hammersmith Hospital, London, UK. Egerton has received consultancy fees from Heptares Therapeutics Ltd and worked on research funded by Hoffman la Roche. Howes has received unrestricted investigator-led charitable funding from or spoken at meetings organized by Astra-Zeneca, Bristol-Myers Squibb, Janssen, Hoffman la Roche, Leyden-Delta and Eli Lilly. McGuire has received consultancy fees from Hoffman la Roche and Sunovion. The remaining authors declare no potential conflicts of interests. [fig] [ 11 C: ]-(+)-PHNO BP ND values in the control and stress conditions are provided in the supplementary table S3. Immigrants demonstrated elevated striatal DA release in response to stress compared to nonmigrant Canadians (F = 8.08; df = 1, 52; P = .006; partial eta2 = 0.13) (figure 1). The clinical group effect was significant (F = 3.57; df = 1, 52; P = .04; partial eta 2 = 0.12), as patients with schizophrenia showed higher striatal DA release than CHR and HV (Bonferroni corrected comparisons, P = .05 and .02, respectively). However, the clinical group by immigration status interaction term was nonsignificant. The significant increase in stress-induced DA release in immigrants was present in the AST (F = 8.05; df = 1, 52; P = .006; partial eta 2 = 0.13; figure 1), LST (F = 5.22; df = 1, 52; P = .03; partial eta 2 = 0.09), and SMST (F = 4.17; df = 1, 52; P = .05; partial eta2 = 0.07). Voxel-wise analysis also revealed significant stress-induced striatal DA release in immigrants (supplementary figure S3) with no significant effect in nonimmigrants. [/fig] [fig] Figure 1: (A) Significant effect of immigration on stress induced DA release in the whole striatum and in striatal subdivisions, including AST, LST, and SMST. WS (F = 8.08; df = 1, 52; P = .006), AST (F = 8.05; df = 1, 52; P = .006), LST (F = 5.22; df = 1, 52; P = .03), and SMST (F = 4.17; df = 1, 52; P = .05). (B) Effects of immigration on stress-induced dopamine release in the whole striatum by clinical vulnerability (mean and standard error of the mean, SEM). [/fig] [fig] Figure 2: (A) Significant effect of immigration on DA synthesis capacity in the whole striatum and SMST, with similar trends in the AST. WS (F = 4.95; df = 1, 73; P = .03), SMST (F = 9.24; df = 1, 73; P = .003), and AST (F = 3.28; df = 1, 73; P = .07). (B) Effects of immigration on DA synthesis capacity in the whole striatum by clinical vulnerability (mean and standard error of the mean, SEM). [/fig] [table] Table 1: Demographic Data by Immigration Status for the Stress-Induced DA Release Study, Canada Site [/table] [table] Table 2: Demographic Data by Immigration Status for the DA Synthesis Study, UK Site [/table]

Development of Walking indicators to advance the quality of spinal cord injury rehabilitation: SCI-High Project Objective: To describe the development of structure, process and outcome indicators that will advance the quality of walking rehabilitation for Canadians with spinal cord injury or disease (SCI/D) by 2020. Method: A framework for the evaluation of the quality of walking rehabilitation was developed by experts in walking after SCI/D. A systematic literature review identified factors influencing walking outcomes and potential walking indicators. A Driver diagram analysis summarized the factors affecting walking outcomes and subsequently informed the selection of structure and process indicators. Psychometric properties and clinical utility of potential walking indicators were considered during the selection of outcome indicators. Results: The structure indicator is the number of physical therapists using evidence-based walking interventions per number of ambulatory individuals with SCI/D. The process indicator is the number of received hours of walking interventions during inpatient rehabilitation per number of ambulatory individuals with SCI/D. The intermediary outcome indicator, which is collected at discharge from inpatient rehabilitation, is either the modified Timed Up and Go or the 10-Meter Walk Test, the choice of measure is dictated by the stage of walking recovery, as defined by the Standing and Walking Assessment Tool. The final outcome indicator, collected at 18 months post-discharge, is the Spinal Cord Independence Measure III-Mobility subscale. Conclusion: The selected indicators align with current clinical practice in Canada. The indicators will direct the timing and enhance the volume of walking therapy delivered, to ultimately increase the proportion of patients who achieve their walking potential by 18 months post-rehabilitation. # Introduction Regaining the ability to walk is a prime concern for individuals living with spinal cord injury or disease (SCI/ D), especially among those with incomplete injuries. [bib_ref] Targeting recovery: priorities of the spinal cordinjured population, Anderson [/bib_ref] [bib_ref] Who wants to walk? Preferences for recovery after SCI: a longitudinal and..., Ditunno [/bib_ref] Walking is reported to be a higher priority among individuals early after SCI (i.e. <2 years post-injury) than among those with more chronic injuries (i.e. ≥5 years post-injury). [bib_ref] Who wants to walk? Preferences for recovery after SCI: a longitudinal and..., Ditunno [/bib_ref] The likelihood of return to walking after SCI is largely dependent upon injury severity (neurological level of injury (NLI) and American Spinal Injury Association Impairment Scale (AIS) grade). [bib_ref] Clinical diagnosis and prognosis following spinal cord injury, Burns [/bib_ref] [bib_ref] Neurologic recovery after traumatic spinal cord injury: data from the Model Spinal..., Marino [/bib_ref] [bib_ref] Who is going to walk? A review of the factors influencing walking..., Scivoletto [/bib_ref] Return to walking is a realistic goal for those with motor incomplete SCI (AIS grades C and D and lower extremity motor score (LEMS) more than 20), [bib_ref] International standards for neurological classification of spinal cord injury (revised, Kirshblum [/bib_ref] as up to 75% of these individuals will regain some walking ability in the first year after injury. [bib_ref] Clinical diagnosis and prognosis following spinal cord injury, Burns [/bib_ref] Conversely, the proportion who regain walking is much smaller among those with motor complete injury (i.e. AIS A and B); 20-50% of individuals diagnosed with AIS B injuries in acute care walk to some extent at one-year post-injury, while those with AIS A injuries rarely do. [bib_ref] Clinical diagnosis and prognosis following spinal cord injury, Burns [/bib_ref] Early intervention to promote the recovery of walking is desirable to leverage the heightened potential for neuroplasticity [bib_ref] Principles of experience-dependent neural plasticity: implications for rehabilitation after brain damage, Kleim [/bib_ref] [bib_ref] Plasticity after spinal cord injury: relevance to recovery and approaches to facilitate..., Onifer [/bib_ref] and augment the natural recovery that occurs during the first year postinjury. [bib_ref] Motor and sensory recovery following incomplete paraplegia, Waters [/bib_ref] With respect to walking, recovery is greatest during the first 9-12 months after injury onset and plateaus at around 12-18 months. [bib_ref] Clinical diagnosis and prognosis following spinal cord injury, Burns [/bib_ref] Given that the mean duration of inpatient rehabilitation is 78 days (IQR 64 days) in Canada, [bib_ref] Moving from prioritization to implementation: the spinal cord injury rehabilitation care high..., Craven [/bib_ref] this is the optimal time for individuals with SCI to participate in individually customized walking training regimens. Presently, walking rehabilitation after SCI/D is focused on those with the greatest potential to walk. A large multi-site study in the United States reported that 36% of inpatients with SCI participated in interventions targeting walking. [bib_ref] The SCIRehab project: treatment time spent in SCI rehabilitation. Physical therapy treatment..., Taylor-Schroeder [/bib_ref] A majority (96%) of whom had AIS D impairment received, on average, 7.5 ± 6.1 h of walking training during their inpatient stay. [bib_ref] The SCIRehab project: treatment time spent in SCI rehabilitation. Physical therapy treatment..., Taylor-Schroeder [/bib_ref] The mean time spent on walking was 60 min per week; this training time exceeded the mean time spent on other activities, such as bed mobility, stretching, strengthening and transfers. [bib_ref] The SCIRehab project: treatment time spent in SCI rehabilitation. Physical therapy treatment..., Taylor-Schroeder [/bib_ref] In Canada, interventions targeting walking have similarly focused on inpatients with SCI/D who are ambulatory (i.e. AIS D) or have the potential to become ambulatory. These individuals were observed to engage in high-intensity activities during inpatient physical therapy, such as walking and leg cycling. [bib_ref] Cardiovascular Stress during inpatient Spinal Cord injury rehabilitation, Zbogar [/bib_ref] In a single physical therapy session, participants took a median of 51 steps (interquartile range: 0-176 steps) at admission to inpatient rehabilitation, and this volume of training increased to a median of 115 steps (interquartile range: 21-313 steps) by discharge. [bib_ref] Movement repetitions in physical and occupational therapy during spinal cord injury rehabilitation, Zbogar [/bib_ref] Metrics such as the duration of walking training (minutes) and the number of steps per training session facilitate evaluation of therapy intensity or volume. Despite the potential clinical utility of these metrics, the number of steps or the duration of walking training are not currently collected using systematic, standardized methods in Canadian SCI/D rehabilitation clinical settings; although they are routinely used in research settings in Alberta, British Colombia, Ontario, and Quebec. In addition to the aforementioned therapy metrics, psychometrically sound measures of walking are important for accurate monitoring and intervention selection during inpatient SCI/D rehabilitation, and monitoring individuals with SCI/D as they transition to outpatient and/or community rehabilitation services. Incorporation of these measures supports the notion that service delivery models should be data-driven, and not dictated by inpatient rehabilitation length of stay. [bib_ref] Predicting rehabilitation length of stay in Canada: it's not just about impairment, Craven [/bib_ref] Walking assessment in SCI rehabilitation and related research settings has focused on timed measures of walking, such as the 6-minute walk test (6MWT) and 10-meter walk test (10MWT). [bib_ref] Outcome measures for gait and ambulation in the spinal cord injury population, Jackson [/bib_ref] [bib_ref] A systematic review of functional ambulation outcome measures in spinal cord injury, Lam [/bib_ref] The 6MWT measures the distance walked overground in six minutes, while the 10MWT measures the time taken to walk 10 m overground. In Canada, the 6MWT and 10MWT are two walking measures included in the Standing and Walking Assessment Tool (SWAT).The SWAT has been implemented at a national level through the Rick Hansen SCI Registry (RHSCIR)as current best practice for walking assessment during inpatient SCI rehabilitation. The SWAT was developed to address the need for a standardized approach to the assessment of walking during inpatient SCI rehabilitation.The SWAT is a staging tool routinely completed at rehabilitation admission and discharge, or when a patient changes stage on the SWAT; whereby, a patient's stage of walking recovery is used to inform the selection of walking measures to describe and evaluate walking ability during inpatient rehabilitation.The SWAT data elements are included in RHSCIR 2.0 and 3.0, enabling site-specific and national-level evaluation of walking outcomes after SCI. [bib_ref] Physical Therapists' Experiences With the SCI Standing and Walking Assessment Toolkit: An..., Musselman [/bib_ref] Implementation of SWAT in clinical practice, however, has been incomplete, [bib_ref] Physical Therapists' Experiences With the SCI Standing and Walking Assessment Toolkit: An..., Musselman [/bib_ref] and further attention is required to support the implementation of walking assessment during inpatient SCI/D rehabilitation. The Spinal Cord Injury Rehabilitation Care High Performance Indicators (SCI-High) Project provided an opportunity to address a priority domain of rehabilitation care related to walking during and following inpatient SCI/D rehabilitation. The overarching aim of the SCI-High Project is to advance SCI/D rehabilitation care, both inpatient and outpatient, for Canadians in the first 18 months after inpatient rehabilitation admission by the year 2020 through the development and implementation of indicators prioritized by clinicians, researchers and individuals living with chronic SCI/D. Quality of care indicators are widely used to identify trends, inform priority setting and policy formulation, and monitor rehabilitation programs and care processes at macro (health system), meso (organization) and micro ( patient) levels. Indicators can measure the structure, process or outcome of health care services and their evaluation can facilitate the sustainability of a high-quality health care delivery system that is based on evidence-informed programs and services. [bib_ref] Impact of benchmarking and clinical decision making tools on rehabilitation length of..., Burns [/bib_ref] Structure indicators are defined by the properties of the setting in which the health care services occur 23 while a process indicator describes the specific activities in providing and receiving of care. [bib_ref] Risk-adjusted mortality rates as a potential outcome indicator for outpatient quality assessments, Selim [/bib_ref] Finally, an outcome indicator evaluates health improvements (or deterioration) attributable to the health care or therapy provided, such as mortality, morbidity, health status, healthrelated quality of life and patient/family/provider satisfaction as a result of the care. [bib_ref] Risk-adjusted mortality rates as a potential outcome indicator for outpatient quality assessments, Selim [/bib_ref] Indicator data can inform comparisons across different health care settings and systems to ensure continuous quality improvement and the establishment of benchmarks for superior organizations. [bib_ref] Developing evidence-based clinical indicators: a state of the art methods primer, Mainz [/bib_ref] This paper describes the context and approach to the development of the Walking indicators for application during the first 18 months after SCI/D rehabilitation admission. The Working Group's objective was to establish a comprehensive framework of structure, process and outcome indicators specific to the domain construct of Walking for implementation in the Canadian rehabilitation health system environment. # Methods The SCI-High Project aims to select, implement and evaluate indicators of quality care for 11 domains of SCI rehabilitation in Canada by 2020. A detailed description of the overall SCI-High Project methods and process for identifying Walking as a priority domain for SCI/D rehabilitation care are described in related manuscripts in this issue. [bib_ref] Prioritization of rehabilitation domains for establishing spinal cord injury high performance indicators..., Alavinia [/bib_ref] [bib_ref] Methods for development of structure, process and outcome indicators for prioritized spinal..., Craven [/bib_ref] In addition to the Project Team (www.sci-high.ca), an External Advisory Committee, National Data Strategy Committee, and a local quality committee consisting of spinal cord rehabilitation leadership supported the global project goals and provided oversight regarding the context for pilot implementation of planned indicators. In brief, the approach to developing the Walking Domain structure, process and outcome indicators followed a modified, but substantially similar, approach to that described by Mainz et al., [bib_ref] Developing evidence-based clinical indicators: a state of the art methods primer, Mainz [/bib_ref] which included the following planning and development phases: (a) formation and organization of the national and local Working Groups; (b) defining and refining the key domain and specific target construct; (c) providing an overview or summary of existing evidence and practice; (d) developing and interpreting a Driver diagram; (e) selecting indicators; and (f ) pilot testing and refinement of the domain-specific structure, process and outcome indicators. Throughout these processes, a facilitated discussion occurred amongst the domain-specific Working Group and the SCI-High Project Team to utilize relevant expertise on the topic, while ensuring the broader goals of the SCI-High Project were aligned across the other 10 domain Working Groups (as appropriate). Experts in walking and relevant stakeholders were invited to participate in the SCI-High Project as members of the domain-specific Working Group based on their practical or empirical knowledge of SCI/D rehabilitation, walking, and patient care. The group was composed of physiotherapists, physiatrists, rehabilitation scientists and postdoctoral fellows. Four meetings of the Walking Working Group were held between February 2016 and June 2017 via conference call, totaling four hours of discussion when developing the indicators, and for an additional two hours thereafter, to refine the indicators and discuss manuscript preparation. The efforts of this Working Group were bootstrapped by the existing SWAT Working Group and their clinical and research activities. [bib_ref] Assessment of upright mobility after spinal cord injury: a "howto" guide to..., Musselman [/bib_ref] In addition, individual members of the Working Group completed their own independent review of the prepared materials, shared resources and/or practice standards, with one another, or conducted independent evaluations of the proposed indicators via email or teleconference outside of the formal scheduled meetings. The Working Groups were asked to develop/select at least one structure, process and outcome indicator related to the Walking Domain. The Project Team stipulated that the indicators must be relevant, concise (10 min or less to implement), feasible, and aligned across the structure, process and outcome indicator in achieving a single substantive advance in SCI/D rehabilitation care. The indicators could be measured using established or new measurement tools (i.e. questionnaires, data collection sheets, laboratory exams, medical record data), depending on the requirements and feasibility of a given indicator. ## Domain-specific working group meetings A national Working Group of subject matter experts was assembled to identify quality indicators for NO. S1 S121 walking rehabilitation after SCI/D. The meetings were organized to frame the context for clinical decision making using an, "eliminate and concentrate" decision-making approach to review the key constructs related to Walking, review available outcome indicators and develop an evidence-informed Driver diagram to establish three indicators of the quality of walking rehabilitation for implementation from the time of rehabilitation admission to 18 months post-injury. Initially, the Working Group used a systematic method that combined scientific evidence and expert consensus for developing a construct definition for walking after SCI/D. In addition, a small number of fundamental and strategically important indicators for the construct of Walking, which were deemed feasible to collect at a clinical level, were selected via consensus. The selection of structure, process and outcome indicators involved a literature search followed by consideration of the factors affecting walking rehabilitation using an Ishikawa (fishbone) or Driver diagram. 30 ## Literature search A comprehensive literature search using MEDLINE, CINAHL and EMBASE applying the MeSH terms "walking" and "spinal cord injury" was conducted by two members of the Project Team independently (MA, MO). The search aimed to identify potential outcome indicators for Walking, as well as the factors that influence walking outcomes in SCI/D rehabilitation. Non-English manuscripts and inappropriate study designs were excluded. ## Driver diagram Following the literature search, the challenge of selecting quality indicators was facilitated by creation of a Driver diagram, which has been reported to enable a root cause analysis of a given problem or situation. [bib_ref] Change management tools part 1: using analysis to investigate problems, Phillips [/bib_ref] As part of the analysis, a graphic illustration was constructed to convey the relationships between SCI/D rehabilitation care related to walking, and factors that influence walking outcomes. Following review of the literature search results, the Walking Working Group engaged in a mind-mapping exercise to identify the factors that affect walking. Subsequently, the identified factors were grouped into categories [fig_ref] Figure 1 Walking: Domain Driver diagram [/fig_ref]. Guided by the Driver diagram using RAND/UCLA 31 methodology, the Walking Working Group proposed structure, process and outcome (intermediary and final) indicators for the quality of walking rehabilitation to be pilot tested for feasibility at the Lyndhurst Centre, Toronto Rehabilitation Institute-University Health Network, Toronto Canada. # Results ## Construct definition The following construct definition was developed by the Walking Working Group to guide the selection of quality indicators: Walking is the ability to move forward over ground using voluntary lower limb movement while controlling one's balance in an upright posture. Safe and efficient walking allows individuals to move purposefully from place to place to explore and participate in their external environments, with or without the assistance of others and/or assistive technologiesin other words, to be independent moving about their home and community, and in their life activities. The Walking Working Group further delineated ambulatory individuals with SCI/D as those individuals who were staged at 2A on the SWAT stages: maximum assist for therapeutic walking indoors 17 [fig_ref] Table 1: Canadian SCI Standing and Walking Assessment Toolkit [/fig_ref]. ## Driver diagram Using the comprehensive search strategy, potential outcome indicators were identified [fig_ref] Table 2: List of walking outcome measures [/fig_ref] , as well as factors that influence walking [fig_ref] Figure 1 Walking: Domain Driver diagram [/fig_ref]. Overall, numerous factors influence walking outcomes during SCI/D rehabilitation, with impairment-related factors being the most frequent. The Walking Working Group arranged the factors that influence the outcome of walking into eight categories: Impairment, Muscle Mechanics, Drug Therapy for Spasticity, Training Type, Comorbidity, Psychosocial, Environmental, and Assistive Device. [fig_ref] Figure 1 Walking: Domain Driver diagram [/fig_ref] shows the Driver diagram for walking among individuals with SCI/D within these categories. ## Selected indicators Structure indicator: This indicator reflects the delivery of walking rehabilitation. As suggested by the "Training" backbone in the Driver diagram [fig_ref] Figure 1 Walking: Domain Driver diagram [/fig_ref] , walking training after SCI/D consists of task-specific and repetitive practice. [bib_ref] Training to achieve over ground walking after spinal cord injury: a review..., Yang [/bib_ref] All walking training methods listed on the "Training" branch of the Driver diagram embody these training principles. The literature on walking training after SCI/D, however, does not support one mode of training over another. [bib_ref] Training to achieve over ground walking after spinal cord injury: a review..., Yang [/bib_ref] For this reason, rather than selecting a physical structure or piece of equipment as the structure indicator, the Walking Working Group chose to describe the physical therapy expertise. The selected structure indicator is the number of physical therapists at a given rehabilitation site, whom demonstrate regular use of evidence-based interventions for walking rehabilitation (see "Training" branch, [fig_ref] Figure 1 Walking: Domain Driver diagram [/fig_ref] after SCI/D, per total number of physical therapists participating in walking intervention service provision at each site in a fiscal year. Here 'regular use' means that evidence-based walking interventions are used with all patients with SCI/D who are ambulatory. The structure indicator is to be collected and reported annually. Process indicator: This indicator reflects the appropriateness of the health care received with respect to walking rehabilitation. Given that walking gains are typically dose-dependent (i.e. the greater the amount of walking practice, the greater the gains), 7,37 the process indicator is the total number of hours of evidence-based walking training received. Physical therapists will enter the total number of minutes spent on walking training as part of their daily workload documentation. This indicator is to be calculated (sum of training minutes) for each patient with SCI/D who meet the requirements of SWAT [stage 2a or higher [fig_ref] Table 1: Canadian SCI Standing and Walking Assessment Toolkit [/fig_ref] ] at the time of discharge from inpatient rehabilitation. Integral to the accurate reporting of this quality indicator is a clear guideline of what therapeutic interventions are considered to be walking interventions. The Walking Working Group agreed upon two criteria for therapists to consider when determining whether a therapeutic activity could count as part of the process of walking rehabilitation. First, the therapeutic activity must be undertaken with the goal of walking in mind. Second, the therapeutic activity must involve whole-or part-practice of walking as task-specificity is deemed a training parameter associated with gains in overground walking function. [bib_ref] Principles of experience-dependent neural plasticity: implications for rehabilitation after brain damage, Kleim [/bib_ref] [bib_ref] Training to achieve over ground walking after spinal cord injury: a review..., Yang [/bib_ref] To facilitate the reporting of the process indicator (i.e. minutes spent engaged in walking training), the Walking Working Group adapted the Physical Therapy Taxonomy created by the SCIRehab Project. [bib_ref] SCIRehab Project series: the physical therapy taxonomy, Natale [/bib_ref] The time spent on each of the 19 activities listed in the Taxonomy was used as a measure of intensity by the SCIRehab researchers and clinicians. [bib_ref] SCIRehab Project series: the physical therapy taxonomy, Natale [/bib_ref] For the purposes of the SCI-High process indicator for walking, 12 activities listed in the SCIRehab Taxonomy were removed as they did not involve whole-or part-practice of walking (e.g. bed mobility, wheelchair mobility, range of motion/stretching, airway/respiratory management, education). Other activities were modified to increase the task-specificity for walking. For example, transfers between sitting and standing were deemed important for the task of walking, while other transfer types found in the SCIRehab Taxonomy were excluded. Further, while the SCIRehab Taxonomy included stair-climbing under the activity of Gait, the Walking Working Group chose to list Stairs as an independent activity, as ascending and descending steps or stairs are commonly performed in daily life. [bib_ref] Walking tasks encountered by urbandwelling adults and persons with incomplete spinal cord..., Musselman [/bib_ref] Lastly, the aquatic environment was not listed as an independent activity as it is in the SCIRehab Taxonomy, but rather it was considered as another medium for walking training. Thus, six therapeutic activities were included in the SCI-High taxonomy of walking interventions to support documentation of the process indicator [fig_ref] Table 3: Taxonomy of walking interventions for process indicator tracking [/fig_ref]. ## Intermediary outcome indicators: The literature review revealed multiple walking measures that have been used with the SCI/D population. The list of measures was reduced by identifying those with high clinical utility and strong psychometric properties in individuals with SCI/D. Factors contributing to a measure's clinical 2A Maximum assistant Ability to stand and initiate reciprocal steps through voluntary L/E movement but requires maximal physical assistance (>50% of total effort) of at least one person and may include use of assistive devices and/or orthoses with the exception of bilateral KAFOs. 2B Moderate assistant Ability to stand and initiate reciprocal steps through voluntary L/E movement but requires moderate physical assistance (25-50% of total effort) of one person and may include use of assistive devices and/or orthoses with the exception of the bilateral KAFOs 2C Minimum assistant Ability to stand and initiate reciprocal steps through voluntary L/E movement but requires minimal physical assistance (<25% of total effort) of one person and may include use of assistive devices and/or orthoses with the exception of the bilateral KAFOs. ## Functional walking capacity (outdoors) Patient is starting to ambulate without Therapist Assistance but still requires Gait Aids/ Orthoses. Patient progresses to ambulating in the Community. 3A Supervised household ambulator Ability to ambulate daily using reciprocal steps over ground for short distances (10-100 m) with supervision. Person may use assistive devices and /or orthoses with the exception of bilateral KAFOs. 3B Independent household ambulator Ability to ambulate daily using reciprocal steps over ground for short distances (10-100 m) independently. Person may use assistive devices and/or orthoses with the exception of the bilateral KAFOs. 3C Community ambulator Ability to ambulate daily using reciprocal steps over ground for long distances (>100 m) independently. Person may use assistive devices and/or orthoses with the exception of the bilateral KAFOs. ## 4 Full walking capacity Patient ambulates independently without Therapist Assistance or Gait Aids/Orthoses. Independent Ambulatorability to ambulate full time daily at home and in the community without assistive devices, orthoses, or physical assistance. utility include the time, expense and training required to administer the measure. [bib_ref] The psychometric properties and clinical utility of measures of walking and mobility..., Tyson [/bib_ref] Six measures of walking were identified as having high clinical utility and established psychometric properties in individuals with SCI/D [fig_ref] Table 2: List of walking outcome measures [/fig_ref]. From this list of six measures, the 10MWT (completed at a comfortable speed) and the modified Timed Up and Go (mTUG) were selected as the intermediary outcome indicators. These measures will be completed within the five days prior to discharge from inpatient SCI/D rehabilitation. The 10MWT has been recommended previously for use with individuals with SCI/D by numerous groups of expert clinicians and researchers. [bib_ref] Outcome measures for gait and ambulation in the spinal cord injury population, Jackson [/bib_ref] [bib_ref] Lower extremity outcome measures: considerations for clinical trials in spinal cord injury, Bolliger [/bib_ref] Further, it is a walking measure that has been reported to change in response to inpatient SCI rehabilitation. [bib_ref] Physical activity outside of structured therapy during inpatient spinal cord injury rehabilitation, Zbogar [/bib_ref] The disadvantage of the 10MWT, however, is that it is appropriate only for those individuals with SCI who can walk without the assistance of another person (i.e. stage 3B or greater on the SWAT).Hence the mTUG, [bib_ref] Spinal cord injury functional ambulation profile: a new measure of walking ability, Musselman [/bib_ref] which accounts for the level of physical assistance in the scoring, was included as an intermediary outcome indicator for those individuals unable to walk without the physical assistance of another person (i.e. SWAT stages 2A, 2B, 2C and 3A). Final outcome indicator: The main consideration when selecting the final outcome indicator was the feasibility of completion. Since this quality indicator is evaluated at 18 months post-discharge from inpatient rehabilitation, a measure that can be completed via survey from the patients' home environments was desired. Therefore, the SCIM III Mobility sub-scale was selected because, as a self-report measure, it can be completed over the phone. [bib_ref] Applicability, validation and reproducibility of the Spinal Cord Independence measure version III..., Almeida [/bib_ref] [bib_ref] Development and validation of a self-report version of the Spinal Cord Independence..., Fekete [/bib_ref] [fig_ref] Table 4: Selected structure, process and outcome indicators for the Walking Domain, and the... [/fig_ref] summarizes the selected structure, process and outcome indicators for the Walking Domain. The identified outcome indicators are appropriate for use with individuals who are ambulatory (i.e. SWAT stage 2A or higher). For non-ambulatory individuals (i.e. SWAT stages 1A, 1B and 1C), the wheelchair mobility indicators will be used. [bib_ref] Development of wheeled mobility indicators to advance the quality of spinal cord..., Bayley [/bib_ref] As some individuals with SCI/D will be wheeling and walking, the Walking Working Group consulted with clinicians to define when: (a) wheelchair mobility indicators should be collected, (b) patients had progressed sufficiently to collect indicators for both wheeling and walking, and (c) walking indicators alone are appropriate to collect. The thresholds for which domain indicators should be collected (i.e. walking or wheeling) are based on the SWAT stages [fig_ref] Figure 2: SCI-High Walking and Wheeled Mobility Domain indicator decision tree [/fig_ref]. # Discussion A comprehensive framework of structure, process and outcome indicators for the quality of walking rehabilitation, as measured during inpatient rehabilitation and post-discharge in the community, was developed [fig_ref] Table 4: Selected structure, process and outcome indicators for the Walking Domain, and the... [/fig_ref]. The structure, process and outcome indicators were selected in part due to their likelihood of implementation and sustainability. This emphasis on clinical feasibility was a strength of the approach used by the Walking Working Group and the SCI-High Project Team. The intermediary and final outcome indicators (i.e. 10MWT, mTUG and SCIM III Mobility subscale) are commonly used measures in physical therapy practice in Canada. Workload documentation, albeit not specific to walking interventions, is also familiar to physical therapists working in publicly-funded rehabilitation hospitals and clinics. Implementation of the Walking Domain indicators in clinical practice will be facilitated through The Journal of Spinal Cord Medicine 2019 VOL. 42 NO. S1 S126 standardized timing for collection of the process and outcome indicators using established documentation tools. Implementation may be further facilitated by accessing pre-existing SCI/D networks or communities of practice, such as the Canadian SCI Walking and Standing Module Group, to assist with the dissemination of knowledge concerning the SCI-High Walking Domain indicators. Implementation of the structure, process and outcome indicators for walking will impact Canadian SCI/D rehabilitation practices. Through collection and analysis of the Walking indicator data, rehabilitation administrators will have site-specific metrics concerning the quality of walking rehabilitation that can be compared to national-level benchmarks. This information may be used to modify site-specific practices, as well as inform provincial-level funding policy decisions and advances in care nationally. For example, the Walking indicators will provide objective data concerning the amount of evidence-based walking interventions received by individuals with SCI/D. Short lengths of hospital stay are known to impact the type and intensity of walking interventions delivered in the USA. [bib_ref] Who wants to walk? Preferences for recovery after SCI: a longitudinal and..., Ditunno [/bib_ref] During these short hospital stays, instead of focusing on walking, the frequent priority in rehabilitation was wheeled mobility, a skill which enables sufficient independence for discharge. [bib_ref] Who wants to walk? Preferences for recovery after SCI: a longitudinal and..., Ditunno [/bib_ref] The reader is encouraged to review the related manuscript describing the indicators associated with wheeled mobility. [bib_ref] Development of wheeled mobility indicators to advance the quality of spinal cord..., Bayley [/bib_ref] Likewise, collection and analysis of intermediary and final outcome indicators will provide an objective description of the trajectory of walking recovery after inpatient rehabilitation among ambulatory individuals with SCI/D, providing insight into whether the current duration of inpatient rehabilitation in Canada 10 is optimal or sub-optimal to address walking deficits in this population. Health system barriers, such as length of stay, are difficult to overcome; however, the Walking indicators may provide valuable data that contributes to future policy discussions. Collection of the Walking indicators will be undertaken by physical therapists working within inpatient SCI/D rehabilitation settings, consequently impacting their workload. The burden of the documentation requirements for the Walking indicators has not yet been evaluated; however, based on previous initiatives and literature concerning the use of standardized outcome measures by physical therapists, we may anticipate some barriers to implementation of the Walking indicators. Previous initiatives involving the implementation of outcome measures by physical therapists have identified the following barriers: a perceived lack of confidence in using outcome measures, constraints of the clinical environment (i.e. lack of space, time and measurement tools), and the challenges associated with changing behavior. [bib_ref] Current use and barriers and facilitators for implementation of standardised measures in..., Swinkels [/bib_ref] [bib_ref] Outcome measures in physiotherapy management of patients with stroke: a survey into..., Van Peppen [/bib_ref] Specific to use of the SWAT during inpatient SCI rehabilitation in Canada, the following factors were identified as barriers to implementation: lack of time, unfamiliarity with the tool, lack of workplace policy requiring completion of the SWAT, lack of applicability to patients who are outliers with respect to walking recovery and reluctance to complete in patients with poor gait quality. [bib_ref] Physical Therapists' Experiences With the SCI Standing and Walking Assessment Toolkit: An..., Musselman [/bib_ref] As the indicators of walking rehabilitation quality are implemented at Canadian SCI/D rehabilitation sites, ongoing training sessions and support for inpatient physical therapists will be offered by the SCI-High Project Team to discern when wheeled mobility or Walking indicators should be collected. These sessions will address barriers to implementation through joint problem-solving. Similar targeted knowledge translation activities have resulted in increased use and interpretation of standardized outcome measures by physical therapists in acute care settings. [bib_ref] Breaking Down barriers to the Utilization of standardized Tests and outcome measures..., Mcdonnell [/bib_ref] Selection of the Walking Domain indicators involved consideration of Canadian SCI/D rehabilitation practice context; hence, the generalizability of the framework of structure, process and outcome indicators for Walking to other clinical settings outside Canada may be limited. For example, physical therapy expertise was selected as the structure indicator; however, in other parts of the world, other health care professionals and/or support workers may be the drivers of walking recovery after SCI/D. In addition, the timing of measurement of Walking indicators that is outlined here [fig_ref] Table 4: Selected structure, process and outcome indicators for the Walking Domain, and the... [/fig_ref] also may not align with health care practices outside of Canada, due to varying lengths of hospital stay and access to rehabilitation after SCI/D. Implementation of a similar framework of quality indicators for walking rehabilitation outside of Canada would require modification of the framework to match the local health care practice and health system design. # Conclusion We have described the process for the identification of the Walking Domain quality indicators for inpatient SCI/D rehabilitation in Canada. Structure, process and outcome indicators that reflect, at a high level, the quantity and quality of therapeutic interventions targeting walking have been selected. Standardized timing for documentation of walking interventions and outcomes will be used to optimize rehabilitative care and promote enhanced walking ability after SCI/D. Facilitation of clinician-, site-, and national-level reflection of current practices, coupled with ongoing review of [fig] Figure 1 Walking: Domain Driver diagram. UEMS: Upper-Extremity Motor Score; LEMS: Lower-Extremity Motor Score; NLI: Neurological Level of Injury; AIS: ASIA Impairment Scale; HR: Heart Rate; BP: Blood Pressure; 4-AP: 4-Aminopyridine; FES: Functional Electrical Stimulation; FET: Functional Electrical Training; PSW: Personal Support Worker. Musselman et al. Development of walking indicators to advance the quality of spinal cord injury rehabilitation The Journal of Spinal Cord Medicine 2019 VOL. 42 NO. S1 [/fig] [fig] Figure 2: SCI-High Walking and Wheeled Mobility Domain indicator decision tree. Appropriate indicator data collection in based on the patient's stage of standing and walking recovery ascertained using the Canadian SCI Standing and Walking Assessment Tool. 17 The Figure is intended to help clinicians decide when it is appropriate or not to collect Walking or Wheeled Mobility indicators, or both, based on the individual's stage of standing and walking recovery.Musselman et al. Development of walking indicators to advance the quality of spinal cord injury rehabilitation [/fig] [table] Table 1: Canadian SCI Standing and Walking Assessment Toolkit (SWAT) Stages of Walking Recovery ©RHSCIR Canadian SCI Standing and Walking Module Group on behalf of Rick Hansen Institute, 2014. [/table] [table] Table 2: List of walking outcome measures. [/table] [table] Table 4: Selected structure, process and outcome indicators for the Walking Domain, and the related denominator for calculating the indicator and the time of collection. Fiscal Year; SWAT = Standing and Walking Assessment Tool; mTUG = modified Timed Up and Go Test; 10MWT = 10 Meter Walk Test; SCIM III = Spinal Cord Independence Measure, Version III. [/table] [table] Table 3: Taxonomy of walking interventions for process indicator tracking. [/table]

Relationship Between Diffusion Tensor Imaging (DTI) Findings and Cognition Following Pediatric TBI: A Meta-Analytic Review This study meta-analyzed research examining relationships between diffusion tensor imaging and cognition following pediatric traumatic brain injury (TBI). Data from 14 studies that correlated fractional anisotropy (FA) or apparent diffusion coefficient/mean diffusivity with cognition were analyzed. Short-term (<4 weeks post-TBI) findings were inconsistent, but, in the medium to long term, FA values for numerous large white matter tracts and the whole brain were related to cognition. However, the analyses were limited by the diversity of brain regions and cognitive outcomes that have been examined; all in relatively small samples. Moreover, additional data are needed to investigate the impact of age and injury severity on these findings. differences in the injuries that are sustained and the associated outcomes. These differences are maximal at birth and then reduce with increasing age. For example, unlike body size, the intracranial volume of a 2-year-old is approximately 72% of that of an adult and by adolescence it is approximately 96% . In addition, TBIs that are sustained during childhood can lead to cognitive impairments that may alter a child's developmental trajectory; thereby having a greater impact than an equivalent injury in an adult [bib_ref] Very long-term neuropsychological and behavioral consequences of mild and complicated mild TBI:..., Hessen [/bib_ref]. The current study therefore focuses only on children and adolescents who have sustained a TBI. Several studies have examined the relationship between cognitive functioning and DTI (FA, ADC/MD) following pediatric TBI. However, the number of studies examining the same brain regions using comparable cognitive measures is often small and the associated findings are often inconsistent. For example, correlations between FA values in the splenium (corpus callosum) and measures of attention/information processing range from no/small relationship in some studies [bib_ref] Diffusion tensor imaging detects white matter abnormalities and associated cognitive deficits in..., Adamson [/bib_ref] [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref] to medium or large positive relationship in others [bib_ref] Working memory and corpus callosum microstructural integrity after pediatric traumatic brain injury:..., *treble [/bib_ref] [bib_ref] Longitudinal changes in the corpus callosum following pediatric traumatic brain injury, Wu [/bib_ref]. Moreover, imaging has been conducted in both the short [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref] and long term [bib_ref] Diffusion tensor imaging detects white matter abnormalities and associated cognitive deficits in..., Adamson [/bib_ref] [bib_ref] Working memory and corpus callosum microstructural integrity after pediatric traumatic brain injury:..., *treble [/bib_ref] [bib_ref] Longitudinal changes in the corpus callosum following pediatric traumatic brain injury, Wu [/bib_ref] after TBI; a difference that is very likely to affect the direction of the relationship between cognitive and DTI measures [bib_ref] A systematic review of diffusion tensor imaging findings in sports-related concussion, Gardner [/bib_ref]. In combination, variability in the brain regions that have been examined, the measures that have been used to assess cognition, and the time at which the imaging is performed have made it difficult to determine whether, and to what extent, there is a relationship between DTI and cognitive outcomes. This, in turn, has limited our ability to evaluate the clinical utility of DTI for predicting cognitive outcomes after pediatric TBI. ## Aims The current study provides a meta-analytic review of research that has examined the relationship between DTI measures of white matter integrity (FA, ADC/MD) and cognitive performance in children and adolescents who have sustained a mild, moderate and severe TBIs. A key goal was to determine whether FA and ADC/MD measures obtained from DTI performed in the short term (≤4 weeks) and medium to long term (>4 weeks) predict cognitive outcomes, with a particular emphasis on the white matter tracts that have previously been reported to be most affected by pediatric TBI [bib_ref] Diffusion tensor imaging (DTI) findings following pediatric nonpenetrating TBI: A meta-analysis, Roberts [/bib_ref]. It was expected that better cognitive performance would be related to (a) lower FA and higher ADC/MD in the short term and (b) higher FA and lower ADC/ MD values in the medium to long term. # Methods ## Literature search A comprehensive search of the literature was undertaken in order to identify research that has examined the relationship between DTI measures of white matter integrity and cognitive functioning in children and/or adolescents who had sustained a TBI. Seven electronic databases were searched (search performed December 2014): PubMed, PsycINFO (Ovid), CINAHL (EbscoHost), Scopus, Embase (Elsevier), Informit, and Web of Science. A broad range of search terms were used to cover: cognition/neurocognition/mental processes, TBI/brain injury/brain trauma, pediatric/child/adolescent, and DTI/DWI/diffusion MRI (see Appendix, for the specific search/logic grids). A study was deemed eligible for inclusion if it met the following criteria: (a) the participants were children and/or adolescents (mean age <18) who had sustained a non-penetrating TBI; (b) cognitive functioning was assessed using objective tests (rating scales, parental questionnaires, and measures of health/emotional functioning were all excluded for current purposes); (c) it examined the relationship between DTI (FA, ADC/MD) and cognitive functioning; (d) it reported original data (excludes reviews); (e) the sample size was adequate for calculating correlations (n > 5); (f) it was published in a journal in English (excludes dissertations); and (g) correlations (Pearson r/Spearman rho or exact p-values) were provided between the cognitive and DTI measures. The literature search identified 787 potentially relevant studies (see [fig_ref] Figure 1: Flow of included studies [/fig_ref] for flow chart). Preliminary application of the inclusion criteria to the titles and abstracts of these papers, reduced this number to 210, 149 of which were duplicates. then obtained and the inclusion criteria re-applied. A further 39 papers were excluded on this basis, with another eight reporting multivariate statistics (general linear models, ANOVAs) [bib_ref] Diffusion tensor imaging detects white matter abnormalities and associated cognitive deficits in..., Adamson [/bib_ref] [bib_ref] Pediatric traumatic brain injury: Language outcomes and their relationship to the arcuate..., Liégeois [/bib_ref] [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref] [bib_ref] Working memory and corpus callosum microstructural integrity after pediatric traumatic brain injury:..., *treble [/bib_ref] [bib_ref] Diffusion tensor imaging in the corpus callosum in children after moderate to..., Wilde [/bib_ref] [bib_ref] A primer of neuroimaging analysis in neruorehabilitation outcome research, Wilde [/bib_ref] [bib_ref] Diffusion tensor imaging of the cingulum bundle in children after traumatic brain..., Wilde [/bib_ref] [bib_ref] Neurocognitive and neuroimaging correlates of pediatric traumatic brain injury: A diffusion tensor..., Wozniak [/bib_ref] , rather than univariate correlations. The corresponding authors for these eight studies were subsequently contacted to request the relevant data. Of these, four provided the necessary data in the form of univariate correlations [bib_ref] Diffusion tensor imaging detects white matter abnormalities and associated cognitive deficits in..., Adamson [/bib_ref] [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref] [bib_ref] Working memory and corpus callosum microstructural integrity after pediatric traumatic brain injury:..., *treble [/bib_ref] [bib_ref] Neurocognitive and neuroimaging correlates of pediatric traumatic brain injury: A diffusion tensor..., Wozniak [/bib_ref] and another replied, but was unable to assist [bib_ref] A primer of neuroimaging analysis in neruorehabilitation outcome research, Wilde [/bib_ref] , leaving a total of 16 studies. Finally, all studies were checked for independence because meta-analyses assume that all samples are independent of one another. Three studies by Caeyenberghs and colleagues [bib_ref] Correlations between white matter integrity and motor function in traumatic brain injury..., Caeyenberghs [/bib_ref] were combined and treated as one on this basis, leaving 14 independent studies from which data could be extracted and analyzed. The PRISMA statement [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: The PRISMA Statement, Moher [/bib_ref] , which outlines the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and provides a 27-item checklist, was completed to ensure that the current meta-analysis met these guidelines (see , supplementary material, for checklist).: general cognition, verbal functions, executive functions, concept formation and reasoning, construction, motor function, memory, attention and information processing, and academic achievement (see , supplementary material, for a list of the cognitive tests, the cognitive domain into which they were classified and studies using these tests). Where studies used multiple cognitive tests to assess the same cognitive domain (e.g., memory), the scores from these tests were averaged so that each study provided only one correlation for each domain (r scores transformed to Fisher's Z, averaged and transformed back to an r;. Mean correlations cannot be calculated directly from r; r was therefore transformed to Fisher's Z r and a mean Z r calculated, which was then converted back to r for ease of interpretation. FA and ADC/MD data were analyzed separately, as were DTI data collected in the short and the medium to long term in order to assess changes in both the direction and rate of diffusion over time. There is considerable variation in the intervals that are used to define short and medium to long term within the TBI literature: we used 4 weeks to divide the study findings into short (≤4 weeks) and medium/long term (>4 weeks). ## Data collection and preparation ## Effect size calculation and interpretation Pearson's r correlation coefficients (or Spearman's rho) assessing the relationship between the cognitive and DTI measures were extracted for each region of interest (ROI) and study. Mean weighted effect sizes (r w ) were calculated when multiple studies provided correlations between the same cognitive domain and ROI. The inverse variance was used to weight effect sizes when calculating means. A positive r (or r w ) indicates that higher FA values or lower ADC/MD values were associated with better cognitive performance, with r values of 0.1, 0.3 and 0.5 equating to small, medium, and large effects, respectively [bib_ref] A power primer, Cohen [/bib_ref]. Probability (p) values were calculated to provide a measure of statistical significance, with a p value < .05 indicating that the true relationship between the cognitive and DTI measures in the pediatric TBI population differs significantly from zero. Ninety-five percent confidence intervals (95%CIs) were calculated to indicate the likely range of the true population effect. Calculations were performed using the Comprehensive Meta-Analysis program (CMA, Version 3;. A random-effects model was used because studies varied in terms of a number of methodological variables (e.g., age, TBI severity). Finally, failsafe Ns (N fs ) were calculated to address the issue of publication bias. This statistic provides a hypothetical measure of the number of unpublished studies with an effect size of zero that would need to exist in order to reduce a finding to a trivial effect (r < .1) and, therefore, call the current results into question [bib_ref] A fail-safe N for effect size in meta-analysis, Orwin [/bib_ref]. The larger the N fs , relative to the number of studies contributing to a result, the less likely it is that this number of unpublished studies with null findings would exist. Ideally, all studies would use the same or comparable cognitive tests to assess specific cognitive domains (e.g., memory) and examine the same ROIs. Unfortunately, this was not the case, with very limited overlap in both the cognitive tests that were used and the brain regions that were examined by researchers. While meta-analyses are designed to pool data from multiple studies, it is not possible to determine the extent to which this is possible until all data have been collected. However, the provision of comparable data (r or r w , p, 95% CIs, N fs statistics)-whether from individual or multiple studies-enables the findings in this emerging area to be directly compared and evaluated, which arguably represents an important advance in the research literature. The inferences made throughout this meta-analysis are based on the aforementioned statistics. Specifically, we argue that we can be more confident that there is a relationship between cognitive functioning and white matter integrity-as measured by FA or ADC/MD-in children and adolescents following a TBI if the correlations are medium or larger in size (r > .3), statistically significant (p < .05), and have acceptable N fs statistics (N fs > N studies ). ## Quality of study reporting All studies were evaluated against the "Strengthening the Reporting of Observational Studies in Epidemiology" (STROBE) statement , which contains a list of 32 items that should be detailed in all observational studies; thereby providing a means by which study reporting quality could be evaluated. All studies were individually evaluated to determine whether it provided information for each of the 32 items (present = 1, partial detail = 0.5, absent = 0). The percentage of studies that reported each item was also calculated (partial details excluded in this calculation). # Results ## Participant and study characteristics Altogether, the 14 studies provided data for a total of 358 participants, with many samples being relatively small in size (see [fig_ref] Table 1: Demographic and imaging data [/fig_ref] for summary demographic data). Participants ranged in age from 7-15 years and, of those for whom there was demographic data, the majority were right-handed (88%), Caucasian (52%), or Hispanic (35%) males (67%). Notably, however, handedness and ethnicity were only reported by a limited number of studies (N = 6 and 4, respectively). Most studies were conducted in the United States (see also , supplementary material, for details of individual studies). Glasgow Coma Scale (GCS) scores were only reported for nine studies, with the mean falling in the moderate TBI category (mean = 9.8, SD = 2.9). However, all studies provided descriptive categorical information relating to injury severity, which indicated that most (n = 11) examined samples with mixed severity (mild, moderate and/or severe) and only three investigated patients with mild (n = 2) or severe (n = 1) TBI. Six studies examined moderate to severe TBI in combination with complicated-mild TBI, which was defined by a GCS score of 13-15 together with visible lesions on CT scans. Most studies (n = 11) performed their DTI in the chronic phase after the TBI (mean = 1.4 years, SD = 1.6), with only three studies reporting findings in the short term (<4 weeks). Imaging and cognitive assessments were completed proximally for all studies except [bib_ref] Neurocognitive outcomes and recovery after pediatric TBI: Meta-analytic review of the literature, Babikian [/bib_ref] , which performed imaging after an average of 6 days and cognitive testing after an average of 25 months post-injury. The data from this study were analysed with the other short-term data because DTI was performed acutely. The majority of studies used a 3 Tesla (3T) strength scanner (n = 10), with the remaining four using 1.5T, and most used a Phillips (n = 8) or Siemens (n = 4) brand scanner. The number of directions for DTI acquisition ranged from 3 to 45 (mean = 22, SD = 12), (see [fig_ref] Table 1: Demographic and imaging data [/fig_ref] , supplementary material, for study-specific imaging details: scanner strength, brand, image acquisition details, software used for pre-/post-processing and analysis, ROI identification). Variability in DTI parameters and preprocessing/post-processing/analysis may have contributed to the heterogeneity of findings, but limited overlap and/or incomplete details prevented any analysis of these variables. ## Strobe ratings of quality of study reporting The reporting quality of the included studies varied widely, depending on what was being assessed (mean percent of studies meeting criteria for each item = 58%, SD = 45, range = 0-100%). Of the 30 items that are relevant here, 14 were reported by 86% (n = 12) or more of the studies, indicating acceptable reporting quality for many items. All studies provided sufficient information regarding their study rational and objectives, study design, definition and measurement of variables, sampling strategy, summary outcome data, key results, and generalizability (see , supplementary material, for STROBE summary data). In contrast, many fewer studies provided information about the study location (14%), reasons for non-participation (7%), rationale for their sample size (7%), and how missing data were managed (7%). Moreover, limited information was provided about potential sources of bias (29%) and sample representativeness was difficult to evaluate due to a lack of information about participant selection (14%). Thus, while many key aspects were reported, it is not possible to determine from the available data whether the study participants are broadly representative of children with TBI. ## Cognitive domains and regions of interest As seen in [fig_ref] Table 2: Summary information relating to the cognitive domains and regions of interest assessed... [/fig_ref] , a variety of different cognitive domains and a very large number of ROI (n = 44) were examined, both in the short and medium to long term, using FA and/or ADC/MD. However, most cognitive domains were only examined by single studies, as were most ROIs; with attention/information processing being the only cognitive domain, and the corpus callosum being the only ROI, to be examined more widely (see [fig_ref] Table 2: Summary information relating to the cognitive domains and regions of interest assessed... [/fig_ref] for summary details and , supplementary material, for specific study details). In addition, most studies focussed on FA, rather than ADC/MD, in the medium to long term (FA: n = 10, ADC/MD: n = 3 studies), with many fewer ROIs examined in the short term and none by more than one study (FA: n = 2, ADC/MD: n = 2 studies). Above all, [fig_ref] Table 2: Summary information relating to the cognitive domains and regions of interest assessed... [/fig_ref] highlights the breadth of the existing research and available data-in terms of cognitive domains, ROIs, DTI measures (FA vs. ADC/MD) and timing (short vs. medium to long term)-which is at the expense of depth (overlap), consequently the current findings should be viewed as preliminary, providing an empirical basis by which to focus future research. ## Relationship between dti and cognition after tbi Overall, there were 31 noteworthy findings across a range of cognitive functions, as indicated by moderate-to-large (r < −.3 or r > .3) and significant (p < .05) correlations that were unlikely to be undermined by any publication bias (N fs > N studies ). [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref] [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref] , the direction of the relationship between FA or ADC/MD and cognition for the various ROIs [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref] -in both the short and medium to long term-was consistent with expectations (short term: negative r between FA and cognition, positive r between ADC and cognition; medium to long term: positive r between FA and cognition, negative r between ADC/MD and cognition). Moreover, in total, there were 64 medium to large effects, 61 of which were in the predicted direction. However, only 48% were statistically significant, probably due to the small sample sizes. Given the large number of findings that needed to be consolidated-combined with the complexity caused by the assessment of FA or ADC/MD in both the short and medium to long term, and the examination of nine different cognitive domains and 44 ROIs-only the most noteworthy (r < −.3 or r > .3, p < .05, N fs > N studies ) are summarized as follows: (1) Contrary to prediction, better concept formation was predicted by higher ADC/MD (short term) in the corpus callosum (body) [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref]. (2) Better executive function was predicted by lower FA (short term) in the cerebral peduncle and internal capsule [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref]. (3) Better memory was predicted by higher ADC (short term) in the left cingulate [fig_ref] Table 3: Pearson FA = fractional anisotropy [/fig_ref] and, higher FA (medium to long term) in the left cingulate and right uncinate fasciculus (1) Medium-long term (>4 weeks) academic achievement (2) attention/information processing (7) construction (1) executive functions (2) general cognition (2) memory (2) motor function (2) Anterior corona radiata (1) Anterior limb internal capsule (3) Brainstem (1) Cerebellum (1) Cerebral peduncle (2) Cingulate gyrus (1) Composite whole brain (2) and lower ADC/MD (medium to long term) in the left frontal lobe and left uncinate fasciculus . (4) Better academic achievement was predicted by higher FA (medium to long term) of the corpus callosum (splenium, isthmus) [fig_ref] Table 4: Pearson r effect sizes [/fig_ref]. (5) Better attention and information processing was predicted by higher FA (medium to long term) in the composite/whole brain, corpus callosum (total, splenium, isthmus) [fig_ref] Table 4: Pearson r effect sizes [/fig_ref] ; and lower ADC/MD (medium to long term) in the left frontal lobe and left cingulate . (6) Better construction was predicted by higher FA (medium to long term) in the corpus callosum (genu-anterior) and lower ADC/MD (medium to long term) in the frontal lobes (left and right) and corpus callosum (anterior) . (7) Better general cognition was predicted by higher FA (medium to long term) of the corpus callosum (isthmus) [fig_ref] Table 4: Pearson r effect sizes [/fig_ref]. (8) Better motor function was predicted by higher FA (medium to long term) in the optic radiation, cortico-spinal tract, posterior thalamic radiation, composite whole brain, medial lemniscus, cerebellum, superior peduncle, corpus callosum (splenium, isthmus, and body) [fig_ref] Table 4: Pearson r effect sizes [/fig_ref]. To summarize, in the short term, contrary to predictions, better concept formation was significantly related with higher FA in the corpus callosum (body). However, consistent with predictions, Moderate-to-large effect sizes-which, when rounded to 1 decimal place to compare against [bib_ref] A power primer, Cohen [/bib_ref] benchmarks, are <−.3 or >.3-only provided here. See , supplementary material, for a complete set of results (small, medium, and large effects). *p < .05. **p < .01. . Pearson r effect sizes (moderate-to-large only) measuring the relationship between ADC/MD and cognitive function in specific ROI in the medium to long term (>4 weeks) post-TBI. a a Moderate-to-large effect sizes-which, when rounded to 1 decimal place to compare against [bib_ref] A power primer, Cohen [/bib_ref] benchmarks, are <−.3 or >.3-only provided here. See material, for a complete set of results (small, medium & large effects). ADC/MD = apparent diffusion coefficient/mean diffusivity; ROI = regions of interest; TBI = traumatic brain injury. ## Cognitive domain *p < .05. **p < .01. better executive function was significantly related with lower FA in two white matter tracts-the cerebral peduncle and internal capsule. Noteworthy findings for ADC in the short term were also contrary to prediction, with better memory being related to higher ADC in the left cingulate. In the medium to long term, better cognitive functioning was consistently related to higher FA values. More specifically, attention/information processing, academic achievement, construction, and general cognition were linked to higher FA values in the corpus callosum. Memory functions were more related to other large white matter tracts, such as the cingulate and uncinate fasciculus, and attention/information processing was additionally related to composite/whole brain FA. In addition, motor functions were consistently related to higher FA values across a range of large white matter tracts and the composite/whole brain. Finally, in the medium to long term, better cognitive function was related to lower ADC/MD values in several ROIs. More particularly, attention/information processing, construction and memory were all related to ADC/MD in the frontal lobes; construction was related to ADC/MD in the corpus callosum; and memory was related to ADC/MD in the cingulate. [bib_ref] Advanced neuroimaging techniques in children with traumatic brain injury, Ashwal [/bib_ref] have suggested that newer imaging techniques, such as DTI, may provide a more sensitive marker of white matter injury than conventional MRI and CT; proposing that they may also better predict cognitive impairments following TBI. This study is the first to systematically synthesize the data from studies that have examined the relationship between DTI findings and tests of cognitive function in children with TBI in order to evaluate the predictive value of DTI. It is also the first study to examine the extent to which these changes vary between the short and medium to long term. # Discussion We analyzed data from 14 studies that correlated FA or ADC/MD with measures of cognitive functioning; the latter being grouped into one of nine broad cognitive domains (general cognition, verbal functions/language skills, executive, concept formation and reasoning, construction, motor function, memory, attention/information processing, academic achievement). In all, the data were collected from 358 participants (229 males, 112 females), with a mean age of 13.5 years (SD = 1.9). One of the most striking findings of this meta-analysis is that there was very limited overlap in the cognitive domains and brain regions (ROIs) that were examined by these 14 studies. In addition, most studies focused on the medium to long term after a TBI, with only three studies assessing the relationship between DTI and cognition performed in the short term. There was also significant heterogeneity in terms of the severity of brain injury, with most studies including participants that had mild or moderate to severe TBIs, without providing data for these subgroups. This variation, combined with the small samples, suggests that the current findings should be viewed as preliminary. Arguably, we can place the greatest confidence in those correlations (between FA or ADC/MD and cognitive functioning) that were medium to large in size (r < −.3 or >.3), statistically significant (p < .05) and had acceptable N fs statistics (N fs > N studies ); consequently the discussion will be confined to these findings. In the first 4 weeks (short term) after a TBI, the findings were not consistent, with both positive and negative relationships reported between FA and cognitive function. In the short term after TBI, and contrary to prediction, we found a strong positive relationship between FA in the corpus callosum (body) and concept formation, with the relationship accounting for over 56% of the variance. This finding contrasted with the large negative relationship between FA in both the cerebral peduncle and internal capsule and executive function, which accounted for between 30% and 36% of the variance. It is not clear why the direction of these relationships differ, but it may be related to the fact that the contributing study [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref] examined children with mild TBIs 2 weeks post-injury; at which time there may be some early resolution of cognitive and physiological changes [bib_ref] Systematic review of the clinical course, natural history, and prognosis for pediatric..., Hung [/bib_ref]. This is also consistent with the finding by this same study that their TBI and healthy controls performed comparably in these cognitive domains. However, it is equally important to note that these results were based on a single small-scale study (N = 16); consequently, they await replication. There were also inconsistencies in the relationship between cognition and ADC/MD in the short term. Although memory was positively related to ADC of the left cingulate, as predicted, all other correlations between cognition and ADC were negative. Once again, these differences may reflect the timing of the DTI, with the former resulting from imaging performed on a mild TBI sample after a mean of three days post-injury (range 1-6 days) [bib_ref] Longitudinal changes in the corpus callosum following pediatric traumatic brain injury, Wu [/bib_ref] and the latter being from a mild, moderate and severe TBI sample that was scanned after a mean of 6 days (range 1-16 days) . Thus, the findings from DTI performed within the first 4 weeks after a TBI are mixed and suggest that additional data are needed to draw firm conclusions about the direction of the relationship between DTI and cognition. Specifically, a finer-grained analysis of the changes that occur during this first month (i.e., comparing acute and post-acute) is now needed, ideally using data from studies that have performed DTI on multiple occasions with the same sample. Based on the available data, the corpus callosum, cerebral peduncle, and internal capsule would appear to be the most promising white matter tracts for continued research, as are complex cognitive functions, such as concept formation and executive functioning. Turning to the medium to long term, there were many more studies that examined the relationship between DTI and cognitive functioning in the months and years after pediatric TBI. Consistent with this, more regions and cognitive domains were examined, but the findings consistently showed that higher FA and lower ADC/MD was associated with better cognitive performance. In fact, there were medium to large and significant correlations between FA or ADC/MD and most cognitive domains. Specifically, cognition (academic achievement, attention/information processing, construction, general cognition, memory) was positively related with FA in one or more of the following regions: the whole brain, corpus callosum, uncinate fasciculus, and cingulate. Thus, better cognition was related to higher FA values, reflecting more uniform water diffusion due to intact white matter tracts. Moreover, better fine motor functioning was related to higher FA in a large number of regions, including the whole brain composite, which is consistent with [bib_ref] A primer of neuroimaging analysis in neruorehabilitation outcome research, Wilde [/bib_ref] suggestion that DTI measures of global white matter may provide the best predictor of cognitive outcomes. However, it is unclear whether this holds true for the other cognitive outcomes, as composite brain measures were only reported for attention/information processing and motor function. Although the findings consistently indicate that there is a positive relationship between cognitive functioning and intact white matter (higher FA) post-TBI, it is important to note that there were only three instances where more than two studies, with over 100 participants (n = 141-164), examined the same cognitive function and ROI (attention/information processing and the splenium, body and genu of the corpus callosum). Thus, given the limited overlap between studies, our conclusions are largely based on data obtained from one to two studies and, frequently, small samples (N = 9-56). Finally, many fewer studies examined the relationship between ADC/MD and cognition in the medium to long term (N = 3), with the most noteworthy findings consistently indicating a negative relationship. That is, consistent with predictions, lower ADC/MD values in the frontal lobe, corpus callosum and cingulate-reflecting less diffusion and intact fibers-predicted better cognitive function. In particular, medium to large and significant relationships were found between: attention/ information processing and ADC/MD in both the left frontal lobe and left cingulate; construction and ADC/MD in both frontal lobes and the anterior corpus callosum; and memory and the left frontal lobe and left uncinate fasciculus. However, caution is again warranted as the findings were largely based on single studies and small samples. In particular, the relationship between attention/ information processing and cognitive function was based on a single small study (N = 6, [bib_ref] Diffusion tensor imaging of incentive effects in prospective memory after pediatric traumatic..., Mccauley [/bib_ref] ; consequently, it needs to be replicated. ## Limitations of the study First, as with all meta-analyses, it is possible that there are studies that were not captured by the current searches. Every attempt was made to minimize the likelihood of this happening by conducting broad searches of seven different data-bases and by calculating N fs statistics, which effectively determine how many "missing" studies (either due to publication bias or failures in the search procedures) would be needed to render a finding inconsequential. Although we did not correct our p values to take into account multiple analyses, we focused solely on moderate to large effects in order to reduce the likelihood of false positives. Second, of the studies that were identified, some failed to provide the required data-either in the original publication or upon written request-and were therefore necessarily excluded from this analysis; thereby limiting the final sample. Third, although STROBE ratings of study reporting quality indicated that many items were reported to an acceptable standard, researchers consistently failed to report a number of aspects of their study design, including participant selection, making it difficult to determine whether the children who were included in this meta-analysis were broadly representative of children who sustain TBIs. Clear details relating to the study recruitment processes and participant retention rates are recommended to address this limitation. Similarly, details of missing data and how these were managed should also be included because it is likely that children who were unable to complete the study were either more severely injured or had pre-injury behavioral or cognitive difficulties, potentially leading to a biased sample. Fourth, studies varied in the pre-processing of data, the software packages that were used to analyse the DTI data, and how ROIs were derived. Different approaches to processing and correctionincluding motion correction, which is a particular issue with pediatric participants-can affect the FA and MD/ADC data, and may have contributed to the heterogeneous findings [bib_ref] Twenty-five pitfalls in the analysis of diffusion MRI data, Jones [/bib_ref]. Unfortunately, there was insufficient data to analyze the impact of these variables on our findings, but these are issues that warrant consideration and highlight the need for studies to provide this information. Finally, the studies examined many different brain regions and used a wide variety of different tests to assess a range of cognitive functions/domains. This meant that, although covering a broad area, there was limited overlap/depth in the research findings. This, in turn, highlights the need for research that uses common measures of cognitive functioning and assesses key ROIs using DTI (e.g., corpus callosum, cingulate, uncinate fasciculus, frontal lobe, whole brain); consistent with a recent recommendation to use common outcome measures in pediatric TBI research . ## Implications for practice and research A recent meta-analysis, which compared the DTI findings of children who had sustained a TBI with healthy controls, identified substantial decreases in FA and/or increases in ADC/MD in the medium to long term following TBI; particularly in the corpus callosum, internal capsule, uncinate fasciculus, longitudinal fasciculus and cingulate, as well as in the frontal, and temporal lobes [bib_ref] Diffusion tensor imaging (DTI) findings following pediatric nonpenetrating TBI: A meta-analysis, Roberts [/bib_ref]. With two exceptions (longitudinal fasciculus, temporal lobes), the current study found that these same ROIs were all related to cognition in the medium-to long-term, suggesting that not only are these regions most affected by TBI, but they are also related to cognitive functioning. Unfortunately, the existing research is largely cross-sectional, with DTI and cognitive function assessed contemporaneously. Clinically, of greatest interest is whether DTI obtained in the acute period predicts longer-term cognition and recovery. Overall, the current findings suggest that there is likely to be significant clinical value in using DTI to predict cognitive functioning, particularly in the medium to long term post-TBI (>4 weeks), by which time there is a consistent relationship between cognitive functioning and FA and ADC/MD values in numerous large white matter tracts, as well as the whole brain. Longitudinal research is therefore now needed to examine whether DTI successfully predicts later cognitive functioning, using those ROIs that the current meta-analysis identified to focus this work. This research should employ designated cognitive tests and focus on those ROIs that show the greatest changes in DTI metrics after pediatric TBI and are also related to cognition when assessed contemporaneously (corpus callosum, internal capsule, uncinate fasciculus, longitudinal fasciculus, cingulate, frontal lobe; [bib_ref] Diffusion tensor imaging (DTI) findings following pediatric nonpenetrating TBI: A meta-analysis, Roberts [/bib_ref]. In addition, age and severity of injury should be considered in order to advance our understanding of how these variables impact on DTI and the relationship between DTI and cognition. In the case of injury severity, preliminary data suggest that the relationship between DTI and cognition may be weak following mild TBI in children [bib_ref] Diffusion abnormalities in pediatric mild traumatic brain injury, Mayer [/bib_ref]. This is broadly consistent with the findings of a recent study of adults, which reported that early white matter changes following mild TBI were no longer evident at 90 days [bib_ref] Multi-modal MRI of mild traumatic brain injury, Narayana [/bib_ref] , suggesting that injury severity and timing of the DTI may be important variables to consider. # Conclusions FA and ADC/MD measures obtained in the medium to long term consistently predict cognitive outcomes assessed contemporaneously following pediatric TBI. Lower FA and higher ADC/MD in the medium to long term-both of which are thought to indicate decreased white matter integrity-predicted poorer performance on a wide range of cognitive functions. DTI of the large white matter tracts-such as the corpus callosum, uncinate fasciculus, and cingulate-and the brain, as a whole, were most predictive of cognitive functioning when using FA. Similarly, DTI of the frontal lobe, uncinate fasciculus, cingulate and corpus callosum were most predictive of cognitive outcomes when using ADC/MD. More research is needed to examine the relationship between DTI and cognition in the short term following pediatric TBI, and to evaluate the potential for DTI performed in the acute period to predict longer-term cognitive outcomes, if it is to be used for clinical purposes. Appendix: Logic grids used in database searches Databases to search: PubMed, PsycINFO, Scopus, Embase, Web of Science, CINAHL, Informit Cognitive function TS = "diffusion tensor imaging" OR TS = dti OR TS = "diffusion magnetic resonance imaging" OR TI = "diffusion tensor imaging" OR TI = dti OR TI = "diffusion magnetic resonance imaging" OR TI = "diffusion weighted imaging" OR TI = dwi OR TS = "diffusion weighted imaging" OR TS = dwi TS = "brain injur*" OR TS = tbi OR TS = "traumatic brain injury" OR TS = "brain trauma" OR TS = "craniocerebral trauma" OR TI = "brain injur*" OR TI = tbi OR TI = "traumatic brain injury" OR TI = "brain trauma" [fig] Figure 1: Flow of included studies. [/fig] [table] Table 1: Demographic and imaging data. [/table] [table] Table 2: Summary information relating to the cognitive domains and regions of interest assessed in the short and medium to long term.Cognitive domains assessed by studies reporting FA (N studies )Brain regions assessed with FA (N studies ) [/table] [table] Table 3: Pearson FA = fractional anisotropy; ROI = regions of interest; TBI = traumatic brain injury.Moderate-to-large effect sizes-which, when rounded to 1 decimal place to compare againstCohen's (1992) benchmarks, are <−.3 or >.3-only provided here. SeeTable G, supplementary material, for a complete set of results (small, medium, and large effects). *p < .05. **p < .01. [/table] [table] Table 4: Pearson r effect sizes (moderate-to-large only) measuring the relationship between FA and cognitive function in specific ROI in the medium to long term (>4 weeks) post-TBI. a [/table] [table] Table A1: Logic grid for PubMed. [/table] [table] Table A3: Logic grid for Scopus. [/table] [table] Table A5: Logic grid for Web of Science. [/table] [table] Table A7: Logic grid for Informit (health databases). SU = Diffusion tensor imaging OR TI,AB = diffusion tensor imaging OR TI,AB = dti OR SU = Diffusion magnetic resonance imaging OR TI, AB = diffusion magnetic resonance imaging) (SU = acquired brain injuries OR TI,AB = tbi OR TI,AB = brain injur* OR SU = traumatic brain injury) (SU = Child OR TI, AB = child* OR SU = Adolescent OR TI, AB = adolesce* OR TI, AB = teen*) (SU = Cognition OR TI, AB = cognition OR TI, AB = cognitive abilit* OR SU = Executive function OR TI, AB = executive function* OR TI, AB = executive control OR SU = mental processes OR TI, AB = mental process*) [/table]

Evaluating the psychometric properties of an e-based version of the 39-item Parkinson’s Disease Questionnaire Background: The 39-item Parkinson's Disease Questionnaire (PDQ-39) is the most thoroughly validated and extensively used self-report measure for the assessment of health-related quality of life in people with Parkinson's (PwP). Given the extent of its use and increasing emphasis on electronic data capture, an e-based version of the PDQ-39, the ePDQ, has recently been developed. The aim of this short report is to present some key reliability and validity data that confirm the psychometric quality of the ePDQ. Findings: Participants were emailed a unique link to an online survey incorporating the ePDQ and demographic questions. A total of 118 PwP fully completed the survey. Floor and ceiling effects were calculated to ensure responses were not biased to extreme values. Consequently, score reliability was assessed by item-total correlations with a range from 0.34 to 0.90. Cronbach's alpha was calculated at between 0.64 and 0.95 for the eight domains of the ePDQ. Construct validity was assessed by comparing domain scores in relation to disease duration and gender, with hypothesised differences being largely confirmed. Construct validity was further assessed following a higher order factor analysis which confirmed the appropriateness of calculating a summary index score. Subsequently, significant, but moderate correlations were calculated between the ePDQ summary index score and disease duration and age at diagnosis. Conclusions: Results indicate that the ePDQ largely mirrors the properties of its parent instrument, the PDQ-39, in terms of reliability and validity. Potential users can therefore incorporate the ePDQ into computer-based data capture systems with confidence. # Introduction The 39-item Parkinson's Disease Questionnaire (PDQ-39) [bib_ref] The development of a short measure of functioning and well being for..., Peto [/bib_ref] is the most thoroughly validated and extensively used self-report measure for the assessment of healthrelated quality of life in people with Parkinson's (PwP) [bib_ref] Health-related quality-of-life scales in Parkinson's disease: critique and recommendations, Martinez-Martin [/bib_ref]. The measure has been shown to possess sound psychometric properties [bib_ref] The development of a short measure of functioning and well being for..., Peto [/bib_ref] [bib_ref] PDQ-39: a review of the development, validation and application of a Parkinson's..., Peto [/bib_ref] [bib_ref] Desirable properties for instruments assessing quality of life: evidence from the PDQ-39, Fitzpatrick [/bib_ref] [bib_ref] Determining minimally important differences for the Parkinson's disease questionnaire (PDQ-39), Peto [/bib_ref] and its use is widely recommended [bib_ref] Health-related quality-of-life scales in Parkinson's disease: critique and recommendations, Martinez-Martin [/bib_ref] [bib_ref] Which clinical measures are most appropriate for measuring disease progression in Parkinson's..., Mcghee [/bib_ref] [bib_ref] Health related quality of life in Parkinson's disease: a systematic review of..., Marinus [/bib_ref]. Given the extent of its use and the increasing emphasis on electronic data capture [bib_ref] Electronic data capture for registries and clinical trials in orthopaedic surgery: open..., Shah [/bib_ref] [bib_ref] Electronic patient-reported outcome systems in oncology clinical practice, Bennett [/bib_ref] [bib_ref] The Computer-based Health Evaluation Software (CHES): a software for electronic patient-reported outcome..., Holzner [/bib_ref] [bib_ref] Validation of electronic systems to collect patient-reported outcome (PRO) data-recommendations for clinical..., Zbrozek [/bib_ref] , an electronic version of the PDQ-39, the ePDQ, has recently been developed. The acceptability and usability of the ePDQ have previously been reported, alongside a study assessing the impact of implementing non-response options versus 'forced response' on response rates and data completeness [bib_ref] An electronic version of the PDQ-39: acceptability to respondents and assessment of..., Morley [/bib_ref]. It has been suggested that full-scale psychometric analysis is not strictly necessary when developing electronic patient reported outcomes (ePROs) which closely mirror their paper-based equivalent [bib_ref] Recommendations on evidence needed to support measurement equivalence between electronic and paper-based..., Coons [/bib_ref]. However, given the extensive use of the PDQ-39, potential users of the electronic version may require reassurance that its psychometric properties are consistent with those of the paper-based version. The aim, therefore, of this short report is to present some key reliability and validity data that confirm the psychometric quality of the ePDQ. # Materials and methods Ethical approval for the study was granted by the Medical Sciences Inter Divisional Research Ethics Committee of the University of Oxford (reference MSD-IDREC-C1-2013-17). ## Participants Recruitment of participants was undertaken with the support of Parkinson's UK. Study details were distributed by the charity's Research Communications Office to their research support network across the United Kingdom. Potential participants were requested to contact the research team by telephone or email. # Materials The ePDQ [bib_ref] An electronic version of the PDQ-39: acceptability to respondents and assessment of..., Morley [/bib_ref] , an electronically administered version of the PDQ-39 [bib_ref] The development of a short measure of functioning and well being for..., Peto [/bib_ref] was administered using Qualtrics survey software. The measure totals 39 items, assessing eight domains of health; Mobility, Activities of Daily Living, Emotional Well-Being, Stigma, Social Support, Cognitions, Communication and Bodily Discomfort. Domain scores are coded on a scale of 0 (perfect health as assessed by the measure) to 100 (worst health as assessed by the measure). A summary index score can subsequently be calculated from the eight domains outlined above [bib_ref] The Parkinson's disease questionnaire (PDQ-39): development and validation of a Parkinson's disease..., Jenkinson [/bib_ref]. Demographic data (gender, age, marital status, ethnic origin and age at or year of diagnosis) were obtained after participants had completed the ePDQ items. ## Procedure After contacting the research team, participants were emailed a unique link to the online survey which could be completed in their own time and at a location of their choice (e.g. home, workplace). A follow-up email was sent after two weeks to non-responders. # Statistical analysis Data was checked for normality of distribution and presence of outliers prior to statistical analysis. Floor and ceiling effects were calculated for each domain of the ePDQ. Reliability was assessed via corrected item-total correlations and Cronbach's alpha. Validity was determined through calculation of differences between known groups using independent samples t-tests. Higher order factor analysis was used to create a summary index score. Relationships between the summary index score and relevant demographic variables were tested via Pearson correlations coefficients in order to further assess validity. # Results A total of 118 PwP (66 males, 52 females) fully completed the ePDQ, a response rate of 91.4%. The mean age was 63.48 years (standard deviation (SD) 8.66), mean disease duration 5.73 years (SD 4.34) and mean age at diagnosis 57.69 years (SD 9.00). ## Reliability Floor and ceiling effects were calculated to ensure responses were not biased to extreme values (see [fig_ref] Table 1: ePDQ item-total correlations with domain floor and ceiling effects and Cronbach's alpha... [/fig_ref]. Consequently score reliability was assessed by item-total correlations (also reported in [fig_ref] Table 1: ePDQ item-total correlations with domain floor and ceiling effects and Cronbach's alpha... [/fig_ref] with a range from 0.34 to 0.90. Cronbach's alpha was calculated at between 0.64 and 0.95 for the eight domains of the ePDQ, indicating sufficient to excellent internal reliability. Alpha values for all domains can again be viewed in [fig_ref] Table 1: ePDQ item-total correlations with domain floor and ceiling effects and Cronbach's alpha... [/fig_ref]. ## Validity Construct validity was assessed by comparing domain scores in relation to disease duration (groups split based on median disease duration of 4 years) and gender, as seen in [fig_ref] Table 2: Comparison of ePDQ domains by disease duration and gender [/fig_ref]. All domains anticipated as being significantly different for disease duration were confirmed as such; Mobility; Activities of Daily Living; Cognitive Impairment; Communication; Bodily Discomfort. Hypothesised nonsignificant differences between males and females were confirmed in all but one of the eight ePDQ domains. Construct validity was further assessed following a higher order factor analysis which confirmed the appropriateness of calculating a summary index score. One factor with an Eigenvalue in excess of one was identified, accounting for 55.4% of variance (extraction method principal component analysis). Each domain of the ePDQ loaded on this one factor which had an eigenvalue of 4.4. Subsequently, all eight domains of the ePDQ were summed to create the summary index score. The mean value of the summary index was 28.67 (S.D. = 16.66, min = 2.97, max = 91.93). Finally, validity was further established by significant, but moderate correlations between the ePDQ summary index score and disease duration (r = .27, p < 0.01) and age at diagnosis (r = −.27, p < 0.01). # Discussion This short report has presented a brief analysis of the newly developed ePDQ as a means of confirming that the measure reflects qualities that are consistent with the paperbased PDQ-39 [bib_ref] The development of a short measure of functioning and well being for..., Peto [/bib_ref]. Floor and ceiling effects generally mirror those of the parent instrument, largely falling within previously used criteria of 20% [bib_ref] Improving the evaluation of therapeutic interventions in multiple sclerosis: development of a..., Hobart [/bib_ref]. It is acknowledged, however, that the domain of Social Support is almost double this figure, which may be a reflection of the inherent weakness of three-item domains. Reliability of the ePDQ is confirmed by two commonly used analyses, item-total correlations and Cronbach's alpha values. All item-total correlations are in excess of previously defined criteria [bib_ref] Advancing the argument for validity of the Alberta Context Tool with healthcare..., Estabrooks [/bib_ref] , the majority significantly so, thereby confirming that item scores within each domain are related to the overall domain score and thus, to the underlying construct. Cronbach's alpha coefficients are above the recommended level of 0.70 in all but one domain, indicating good to excellent internal reliability. The one domain to fall below this figure, Cognitive Impairment at 0.64, remains above the level regarded as sufficient. The validity of the ePDQ was confirmed via a number of analyses. Firstly, a comparison of known groups was made, (sometimes referred to as known groups validity). Such an assessment is made where there are good reasons to hypothesise that scores on a construct being measured will differ between two groups [bib_ref] A checklist for judging preference-based measures of health related quality of life:..., Brazier [/bib_ref] , as has been addressed in previous research [bib_ref] Refinement and validation of the parental illness impact scale, Morley [/bib_ref] [bib_ref] How valid and responsive are generic health status measures, such as EQ-5D..., Papaioannou [/bib_ref]. When comparing domains by disease duration those hypothesised as being significantly different were confirmed (Mobility, Activities of Daily Living, Cognitive Impairment, Communication and Bodily Discomfort). The three domains of Emotional Well-Being, Stigma and Social Support are deemed not to be as sensitive to disease duration with results appearing to support this. Additionally, nonsignificant differences between males and females were observed in seven of the eight ePDQ domains. Construct validity was further supported through the use of a higher order factor analysis to confirm the appropriateness of summing domain scores to create a summary index score. This technique has been widely used in previous research [bib_ref] The Parkinson's disease questionnaire (PDQ-39): development and validation of a Parkinson's disease..., Jenkinson [/bib_ref] [bib_ref] Comparison of methods for scoring and statistical analysis of SF-36 health profile..., Ware [/bib_ref] [bib_ref] The Manchester-Oxford Foot Questionnaire (MOXFQ): development and validation of a summary index..., Morley [/bib_ref] [bib_ref] The PDQ-Carer: development and validation of a summary index score, Morley [/bib_ref]. Summary index scores were found to correlate significantly, and in the anticipated direction, with relevant demographic variables to provide further evidence of validity. It is acknowledged that this study may be limited by the relatively small sample size, although this is adequate for the analyses reported. It is, however, recommended that the ePDQ be further assessed in larger surveys and over time in longitudinal studies. Assessment of the sensitivity of the ePDQ is also required, although given that the reliability and validity of the measure largely mirrors that of the paper-based version, it is anticipated that sensitivity data should follow a similar pattern. Additionally it is acknowledged that the sample reported here may not be entirely representative of the Parkinson's population at large, as not all PwP will have access to electronically administered measures or be computer literate. In conclusion, data indicates that the ePDQ possesses appropriate levels of reliability and validity and can be incorporated into studies with confidence by those who wish to do so. Should readers wish to make direct comparisons between the psychometric properties of the PDQ-39 and ePDQ they can do so via the data presented here and the original validation paper for the PDQ-39 [bib_ref] The development of a short measure of functioning and well being for..., Peto [/bib_ref] or current user manual. Further details of the ePDQ, the PDQ-39 and other related measures, along with how to obtain copies, can be obtained from DM or CJ. Competing interests CJ is a developer of the PDQ-39 and receives royalties from its use. DM, LK and JD have all undertaken consultancy work for ISIS Outcomes who hold the license for the PDQ-39. SD has no conflict of interest to report. Authors' contributions DM contributed to the study design, conducted the statistical analyses and drafted the manuscript. SD collected data and managed the study on a daily basis. LK designed the electronic questionnaire. JD contributed to the study design. CJ developed the study design and was the principal investigator. All authors read and approved the final manuscript. [table] Table 1: ePDQ item-total correlations with domain floor and ceiling effects and Cronbach's alpha values [/table] [table] Table 2: Comparison of ePDQ domains by disease duration and gender(standard deviation; NS = non-significant). [/table]

Ridinilazole, a narrow spectrum antibiotic for treatment of Clostridioides difficile infection, enhances preservation of microbiota-dependent bile acids CM. Ridinilazole, a narrow spectrum antibiotic for treatment of Clostridioides difficile infection, enhances preservation of microbiota-dependent bile acids. # Introduction Clostridioides difficile is a spore-forming organism that can colonize the human intestine. In susceptible individuals, C. difficile infection (CDI) results in significant morbidity and mortality. On average, recurrence of CDI following antibiotic treatment is seen in ϳ20% of cases [bib_ref] Burden of Clostridium difficile infection in the United States, Lessa [/bib_ref] , with increasing rates of recurrence after the first recurrence (reviewed in Ref. [bib_ref] Novel fidaxomicin treatment regimens for patients with multiple clostridium difficile infection recurrences..., Soriano [/bib_ref]. Disruption of the intestinal microbiota by antibiotic treatment is a major risk factor for CDI. An increasing body of evidence suggests that the intestinal bile acid profile plays an important role in both development of disease and resistance to recurrence [bib_ref] A Gut Odyssey: the impact of the microbiota on Clostridium difficile spore..., Shen [/bib_ref] [bib_ref] Impact of microbial derived secondary bile acids on colonization resistance against Clostridium..., Winston [/bib_ref]. Commensal bacteria in the intestine metabolize conjugated bile acids by the activity of two sets of enzymes. Bile salt hydrolases (BSHs) remove the conjugated taurine or glycine to generate unconjugated bile acids. Unconjugated primary bile acids can be further metabolized by the 7␣dehydroxylation pathway to produce secondary bile acids. In vitro, the conjugated and unconjugated primary bile acids taurocholate (TCA) and cholate (CA), respectively, promote C. difficile spore germination, whereas secondary bile acids such as lithocholate (LCA) and deoxycholate (DCA) generally inhibit vegetative growth. However, sporulation and growth are complex processes [bib_ref] Clostridioides difficile biology: sporulation, germination, and corresponding therapies for C. difficile infection, Zhu [/bib_ref] , and different strains of C. difficile exhibit varying responses to bile acids [bib_ref] Inhibition of spore germination, growth, and toxin activity of clinically relevant C...., Thanissery [/bib_ref]. For example, a recent study using C. difficile 630⌬erm showed that both DCA and chenodeoxycholate, a primary bile salt, promote biofilm formation, an effect that is potentiated by fermentable sugars [bib_ref] A microbiota-generated bile salt induces biofilm formation in Clostridium difficile, Dubois [/bib_ref]. Furthermore, the study reports that cells in DCA-induced biofilm show markedly lower sensitivity to vancomycin and metronidazole compared with planktonic cells, whereas DCA exposure enhances the sensitivity of planktonic cells to the antibiotics. Taken together, these studies indicate that bile acids can directly affect C. difficile germination and growth while underlining the complexity of these effects, which vary depending on bile acid and growth environment. The in vitro findings are consistent with studies in mice, which also showed that resistance to C. difficile germination and growth is strongly associated with the presence of specific secondary bile acids [bib_ref] Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to..., Theriot [/bib_ref]. Moreover, the murine studies found that antibiotic treatment leads to a loss in secondary bile acids [bib_ref] Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to..., Theriot [/bib_ref]. In humans, several studies have linked differences in bile acid levels to specific clinical states. These studies generally report higher primary bile acids in recurrent C. difficile patients and higher secondary bile acids in healthy subjects or patients with recurrent CDI who have undergone successful fecal microbiota transplantation (FMT) [bib_ref] Fecal microbiota transplantation via colonoscopy for recurrent C. difficile Infection, Allegretti [/bib_ref] [bib_ref] Changes in microbiota composition, bile and fatty acid metabolism, in successful faecal..., Brown [/bib_ref] [bib_ref] Restoration of short chain fatty acid and bile acid metabolism following fecal..., Seekatz [/bib_ref] [bib_ref] Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile..., Weingarden [/bib_ref] [bib_ref] Changes in colonic bile acid composition following fecal microbiota transplantation are sufficient..., Weingarden [/bib_ref]. Normalization of intestinal microbial community structure in patients undergoing FMT occurs concomitant with restoration of secondary bile acid levels. In the colon, formation of secondary bile acids such as LCA and DCA depends on expression of genes in the bile acid inducible (bai) operon encoding key enzymes of the 7␣-dehydroxylation pathway [bib_ref] Metabolism of hydrogen gases and bile acids in the gut microbiome, Hylemon [/bib_ref]. Unlike genes encoding BSH, which are widely distributed among gut bacteria of different phyla, the bai operon appears to be confined to a relatively small number of species, one of which is Clostridium scindens. An analysis of microbiome data from CDI-diagnosed and C. difficile carrier patients undergoing allogeneic hematopoietic stem cell transplantation found a strong association between the presence of C. scindens and C. difficile colonization resistance [bib_ref] Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile, Buffie [/bib_ref]. Whereas murine model and human FMT studies have shown strong associations between CDI recurrence and intestinal bile acid profiles, relatively little has been reported regarding the effects of different antibiotic treatments on bile acid levels in humans. Previously, bile acid levels in humans with CDI have been measured only at a single time point or before and after an intervention such as FMT. In this study, we characterize the trajectory of stool bile acid levels over time in human subjects diagnosed with CDI enrolled in a phase 2 clinical trial comparing the treatment efficacy of a broad-spectrum agent (vancomycin) against a narrow spectrum investigational agent (ridinilazole) [bib_ref] CoDIFy study group. Efficacy and safety of ridinilazole compared with vancomycin for..., Vickers [/bib_ref]. In the phase 2 trial, treatment with ridinilazole was associated with fewer recurrences. We have previously shown that vancomycin-treated subjects showed dramatic losses of Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae in stool collected at the end of treatment (EOT), with concomitant expansion of species belonging to the phylum proteobacteria [bib_ref] Substituting whole grains for refined grains in a 6-wk randomized trial has..., Thorpe [/bib_ref]. In contrast, ridinilazole-treated subjects showed minimal disturbance in their gut microbiota compared with when the subjects enrolled in the trial (baseline). Based on these observations, we tested the hypothesis that secondary bile acid levels would be depleted in the stool of vancomycin-treated subjects compared with ridinilazoletreated subjects and that the depletion of bile acids would correlate with losses of specific gut bacteria. # Materials and methods # Materials. Glycocholic acid (GCA) and glycodeoxycholic acid (GDCA) were purchased from Cayman Chemical (Ann Arbor, MI). Glycolithocholic acid (GLCA) was purchased from Isosciences (Ambler, PA). Unless otherwise noted, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Participants and sample collection. Stool samples were collected at multiple time points as part of a recent phase 2, double-blind, randomized, controlled, noninferiority clinical trial, as described [bib_ref] Substituting whole grains for refined grains in a 6-wk randomized trial has..., Thorpe [/bib_ref]. Institutional review boards at each enrolling center provided ethics approval, and the study complied with the ethical principles expressed in the Declaration of Helsinki and followed all principles of good clinical practice. Written, informed consent was obtained from all participants. The Tufts Institutional Review Board determined that the present study using deidentified stools constituted nonhuman subject research. In the trial, 100 subjects were enrolled at 33 medical centers in the United States and Canada and randomized at a 1:1 allocation ratio to receive 10 days of either vancomycin or ridinilazole. Partic-ipants were recruited between June 26, 2014, and August 31, 2015. Stool samples were obtained at study entry (baseline), end of treatment [EOT; day 10 (D10)], day 25 (D25), and day 40 postentry (D40) and when a recurrence was suspected. Of the 100 subjects enrolled in the clinical trial, 18 provided fewer than three stool samples for analysis and were eliminated from this study. Of the remaining 82 participants, there were 41 per treatment arm. Because any antibiotic therapy can alter the intestinal microbiota and thus introduce confounding effects, participants who received standard CDI treatments (metronidazole or vancomycin) up to 24 h before randomization and/or who were receiving nonCDI related antibiotic therapy at study enrollment were excluded from this analysis to capture the effects of ridinilazole or vancomycin alone (for consort diagram, see Supplemental ; Supplemental Material for this article can found online at https://doi.org/10.6084/m9.figshare.11859225.v5). This reduced the study groups to 22 subjects per treatment arm. When new antibiotics for either CDI recurrence or a new infection were started, subsequent samples were censored from the analysis, unless otherwise noted. There were no significant differences in age, sex, body mass index (BMI), or use of proton pump inhibitors (PPIs), nonsteroidal anti-inflammatory drugs (NSAIDS), opioids, or probiotics between treatment groups. Control stool samples were obtained from volunteers enrolled in a separate study (44), because the phase 2-only enrolled subjects diagnosed with CDI. The mean ages of the treatment groups and healthy controls are not significantly different. Importantly, the stool samples from all subjects (CDI subjects and healthy control) were handled, stored, processed, and analyzed for microbiota composition and metabolite contents using identical methods. This eliminated potentially confounding influences that could be present due to methodological differences between the two studies. Extraction of metabolites from stool samples. Frozen stool samples stored at Ϫ80°C were lyophilized overnight in 2.0-mL screw-capped tubes (Sarstedt). After recording the dry weight, 0.5 g of 0.1-mm diameter zirconia/silica beads (BioSpec Products) and 1.5 mL of an ice-cold chloroform-methanol (2:1, vol/vol) solution were added to each sample tube. The tubes were then held on a bead-beater (Vortex Genie 2 with MoBio Vortex adapter) for 5 min at 4°C. The debris was pelleted by centrifugation at 16,000 g for 10 min. The supernatant was carefully removed and filtered over a 70-m cell strainer (Thermo Fisher Scientific) into a fresh sample tube. After the addition of ice-cold HPLC-grade water at 60% of the filtrate's volume, the sample tube was vortexed for 20 s and centrifuged at 16,000 g and 4°C for 10 min. The polar layer was then carefully removed using a syringe and dried in a Savant Speed-Vac (Thermo Fisher Scientific) for 5 h. The dried extracts were stored at Ϫ80°C before metabolite analysis. Of note, all stool samples underwent a freeze-thaw cycle before extraction; this did not affect bile acid levels. Targeted analysis of bile acids. The dried samples were reconstituted in 100 L of methanol/water (1:1, vol/vol). Bile acids were quantified using targeted liquid chromatography-mass spectrometry (LC-MS) experiments performed on a triple-quadrupole mass analyzer (QQQ 6410A; Agilent, Santa Clara, CA). A previously described multiple reaction monitoring (MRM) method (33) was modified to improve separation between bile acids of similar mass and chemical structure. Chromatographic separation was achieved on a C18 column (Kinetex 5 m EVO C18 100A, 150 ϫ 2.1 mm; Phenomenex) using a gradient method with two mobile phases (Supplemental [fig_ref] Table 1: Bile acids measured in this study [/fig_ref] , https://doi.org/10.6084/m9.figshare.12217979.v1). Solvent A was methanol-water (1:1, vol/vol) with 10 mM ammonium acetate and 0.1% (wt/vol) ammonium hydroxide (pH 9). Solvent B was methanol with the same concentrations of ammonium acetate and ammonium hydroxide as solvent A (pH 9). The injection volume was 10 L, and column oven temperature was set to 50°C. The mass spectrometer was operated in negative ionization (ESIϪ) mode. The optimized MRM transitions and retention times of chemical standards are shown in Supplemental , https://doi.org/10.6084/m9. figshare.12217979.v1). The MRM scan data were acquired using MassHunter (version B.05.00; Agilent) and imported into Skyline [bib_ref] Skyline: an open source document editor for creating and analyzing targeted proteomics..., Maclean [/bib_ref] for peak identification and integration of areas under the curve (AUCs). Peaks in the ion chromatograms were identified based on retention time (RT) and MRM transition. For quantification of bile acids, concentrations were calculated from the AUCs using standard curves generated from high-purity standards. The bile acid concentrations were normalized by the corresponding sample dry weight. Estimation of BSH gene abundance. The PICRUSt2 software package (https://github.com/picrust/picrust2) was used to estimate the distribution of BSH gene abundances in the stool samples. Metagenome functional content was predicted from 16S rRNA survey data collected in previous studies [bib_ref] Substituting whole grains for refined grains in a 6-wk randomized trial has..., Thorpe [/bib_ref] on the same set of samples analyzed for metabolites in this study. Standard procedures were followed to process the 16S rRNA data using QIIME (10) and generate an operational taxonomic unit (OTU) table as input for PICRUSt2. Briefly, PICRUSt2 uses an evolutionary model and complete sequenced genomes as a reference tree of life to compute an estimate for gene family copy numbers of bacterial types or OTUs. The abundance of each OTU is divided by its predicted 16S rRNA copy number and then multiplied by the copy numbers of gene families. The result is an estimate for each OUT's contribution to a sample's overall gene content or metagenome. Untargeted analysis of stool metabolites. The untargeted LC-MS experiments were performed on the same extracted samples as the targeted experiments on bile acids. The samples were analyzed for global metabolite profiles using information-dependent acquisition (IDA) experiments performed on a triple-quadrupole time-of-flight (TOF) instrument (5600ϩ; AB Sciex) coupled to a binary pump HPLC system (1260 Infinity; Agilent). Each sample was analyzed four times using a combination of positive and negative ionization modes and two different LC methods (Supplemental Methods, https:// doi.org/10.6084/m9.figshare.12217979.v1) to obtain broad coverage of metabolites having varying polarities and isoelectric points. Raw data were preprocessed using XCMS [bib_ref] XCMS-MRM and METLIN-MRM: a cloud library and public resource for targeted analysis..., Domingo-Almenara [/bib_ref] to extract and align peaks. The peaks were then processed using CAMERA [bib_ref] ERA: an integrated strategy for compound spectra extraction and annotation of liquid..., Kuhl [/bib_ref] to identify and remove predicted isotopes, adducts, and in-source fragments. The retained peaks were organized into a feature table, with each feature specified by accurate mass-to-charge ratio (m/z), retention time (RT), and responses representing the AUC for the extracted ion chromatogram of the feature. The AUC of a feature was normalized by the corresponding sample dry weight. In the case that a precursor ion detected by the IDA's survey scan triggered an MS/MS scan, the corresponding MS/MS spectrum was extracted and searched against the spectral libraries of HMDB (54) and NIST17. The MS/MS spectrum of each feature was also analyzed using in silico fragmentation tools MetFrag (55) and CFM-ID (4). These analyses identified several annotations for many of the features. To determine the most likely identities for these features in the context of human intestinal metabolism, we applied an automated annotation procedure ("BioCAn") that combines the outputs from the database searches and fragmentation analyses with a metabolic model for the biological system of interest (1). Briefly, BioCAn maps each unique mass in the feature table onto a metabolic network representing the enzymatic reactions possible in the system of interest and evaluates the likelihood that a correct mapping between a detected mass and a metabolite in the network has occurred based on two factors: how many other metabolites in the neighborhood (defined by reaction distance) of the metabolite in question also map to a detected mass and the relative confidence in the presence of that compound and its neighbors based on results from MS/MS matching to spectral databases and in silico tools (Supplemental Methods). The metabolic network for BioCAn was assembled from the predicted metagenomes obtained using PICRUSt2. A separate network was assembled for annotation of ridinilazole and vancomycin samples to account for the differences in microbiomes between the two treatment groups. The gene functions predicted by PICRUSt2 for ridinilazole and vancomycin samples were tabulated by their KEGG Orthology identifiers (K numbers) and estimated function counts [bib_ref] New approach for understanding genome variations in KEGG, Kanehisa [/bib_ref]. After nonenzymatic functions were eliminated, the K numbers were further filtered to remove functions that have very low counts (median count Ͻ10 across all ridinilazole or vancomycin samples) and are thus unlikely to play a quantitative role in microbiota metabolism. The remaining enzymatic functions were then linked to KEGG reaction identifiers (r numbers) and their primary substrate-product pairs as defined by KEGG's RCLASS data [bib_ref] Reactant pairs and reaction organization patterns produced by a new rule-based approach, Vazquez-Hernandez [/bib_ref]. The substrate-product pairs were used to draw the network graphs for annotation, where the nodes and edges correspond to metabolites and reactions, respectively. Correlation analysis. To determine whether there are significant associations between specific bile acids and bacterial groups, rank correlation coefficients (Spearman's rho) were calculated between OTU counts (relative abundance) and bile acid concentrations. To lessen the chance of detecting spurious correlations, OTUs were excluded when detected in Ͻ5% of the samples, although this may result in missing true correlations with low-abundance organisms. The remaining 1,488 OTUs were grouped by family. In a handful of cases, OTU picking did not resolve the organism at the family level. In these cases, the OTUs were grouped by order. This resulted in a reduced OTU table of 35 families and three orders. Correlations (rho values) were calculated for each bile acid and family/order across samples from ridinilazole-treated subjects, vancomycin-treated subjects, antibiotic-treated CDI subjects (ABX), and all subjects (ALL), including healthy controls. The P values were corrected for false discovery rate (FDR) using the Benjamini-Hochberg (B-H) method [bib_ref] Controlling the false discovery rate -a practical and powerful approach to multiple..., Benjamini [/bib_ref]. A corrected P value of Ͻ0.05 was considered statistically significant. Statistical analysis. Within-group differences in bile acid levels between time points (e.g., ridinilazole baseline vs. ridinilazole EOT) were tested by using a Wilcoxon signed-rank test, and the differences between groups at the same time point (e.g., ridinilazole EOT vs. vancomycin EOT) were analyzed by using a Wilcoxon rank-sum test. A P value of Ͻ0.05 was considered statistically significant. To test whether the changes over time in bile acid composition differed between treatment groups, the relative abundance of each bile acid group (primary, secondary, conjugated primary, or conjugated secondary) at each time point was summarized as an average percentage of total bile acids and analyzed using a linear mixed-effects model (MIXED procedure, SAS 9.4). The model included treatment, time, and their interactions as fixed effects. Bayesian information criterion was used to determine the best-fitting covariance structure, which was first-order autoregressive. We used random forest (Supplemental Methods) to build a classification model for the purpose of identifying individual bile acids and bile acid ratios that can best predict whether the sample was obtained from a subject treated with ridinilazole or vancomycin. For the untargeted metabolites analysis, significance of differences between ridinilazole and vancomycin treatment groups at EOT was determined using Student's t test. The P values from multiple comparisons were corrected for FDR using the B-H method [bib_ref] Controlling the false discovery rate -a practical and powerful approach to multiple..., Benjamini [/bib_ref]. # Results ## Ridinilazole treatment preserves secondary bile acids. We compared the effects of a broad-spectrum antibiotic, vancomycin, which is commonly used to treat CDI, against the effects of a selectively targeting antibiotic, ridinilazole (47), on the bile acids listed in [fig_ref] Table 1: Bile acids measured in this study [/fig_ref]. The concentrations of these bile acids in stool samples from vancomycin-or ridinilazole-treated subjects (in mol/g stool dry wet), as well as in stool samples from a cohort of healthy subjects, are shown in Supplemental [fig_ref] Figure 2: Bile acid [/fig_ref] (https://doi.org/10.6084/m9.figshare.11859228). Bile acids and their groupings that were quantified in this study are shown in [fig_ref] Table 1: Bile acids measured in this study [/fig_ref]. The total concentration of unconjugated primary bile acids (referred to hereafter simply as primary bile acids) trended similarly for both ridinilazole and vancomycin subjects . A significant difference was observed for total conjugated primary bile acids. Subjects receiving ridinilazole showed no significant change from their individual baselines in conjugated primary bile acids throughout the study period . In contrast, subjects receiving vancomycin showed an order-ofmagnitude increase in conjugated primary bile acids at EOT compared with baseline (P Ͻ 0.001). A significant difference between ridinilazole and vancomycin groups was also observed for secondary bile acids, which result from metabolism of unconjugated primary bile acids via a bacterially encoded 7␣-dehydroxylation pathway. Whereas ridinilazole subjects exhibited a stable secondary bile acid profile relative to baseline, vancomycin subjects showed an ϳ10-fold decrease (P Ͻ 0.01) in these bile acids at EOT before they returned to baseline on D25 and D40 . The ratio of total conjugated (primary and secondary) to secondary bile acids was examined. This ratio is the most important classifier of treatment group at EOT, as determined by a random forest model (Supplemental [fig_ref] Figure 3: Bile salt hydrolase [/fig_ref] , https:// figshare.com/articles/Figure_S3/11859246), and is also a measure of both bacterial BSH activity and 7␣-dehydroxylation pathway enzymatic activity on the stool bile acid pool. At baseline, the ratio was not significantly different between the ridinilazole and vancomycin groups (1.5 Ϯ 0.7 and 4.6 Ϯ 4.0, respectively). However, there was an ϳ100-fold increase (P Ͻ 0.0001) in this ratio at EOT relative to baseline for the vancomycin group, whereas the ratio was maintained near baseline for the ridinilazole group . Ridinilazole treatment maintains bile acid composition. We next compared the effect of ridinilazole or vancomycin on the relative abundances of bile acids at different time points. This analysis was undertaken because within the stool matrix, organisms encounter complex mixtures of bile acids. In vitro, combinations of bile acids have been shown to affect C. difficile germination differently than a single bile acid when given alone in the same concentration [bib_ref] Inhibition of spore germination, growth, and toxin activity of clinically relevant C...., Thanissery [/bib_ref]. [fig_ref] Figure 2: Bile acid [/fig_ref] shows the total bile acid concentration [fig_ref] Figure 2: Bile acid [/fig_ref] and relative abundance of each bile acid category (primary, secondary, conju- Fold changes in concentrations of unconjugated primary bile acids (A), conjugated primary bile acids (B), and unconjugated secondary bile acids (C) and ratio of total conjugated (conjugated primary and conjugated secondary bile acids) to unconjugated secondary bile acids (D) were calculated for each subject relative to the subject's baseline, with concentrations measured in mol/g dry stool weight. Data shown are means Ϯ SE. See [fig_ref] Table 1: Bile acids measured in this study [/fig_ref] gated primary, or conjugated secondary) as an average percentage of total bile acids [fig_ref] Figure 2: Bile acid [/fig_ref] , D-F). Differences in changes across time and treatment were assessed using a linear mixedeffects procedure. No significant differences between time points or treatment groups were found for total bile acid. At baseline, vancomycin-and ridinilazole-treated subjects had similar bile acid compositions, with unconjugated (primary and secondary) bile acids accounting for Ͼ90% of the total in both groups. At EOT (D10), vancomycin-treated subjects had a significant increase in the average proportion of conjugated primary bile acids (35%, P Ͻ 0.0001) and a significant decrease in the average proportion of unconjugated secondary bile acids (Ͻ1%, P Ͻ 0.001) compared with ridinilazoletreated subjects. On D40, both ridinilazole-and vancomycintreated subjects had similarly high average proportions of unconjugated bile acids (97% and 94% for vancomycin and ridinilazole, respectively). There was no longer a statistically significant difference in the average proportion of conjugated primary bile acids between the two groups. However, the ridinilazole group still had a significantly greater (P Ͻ 0.01) average proportion of secondary bile acids (73%) compared with the vancomycin group (38%), and there was a statistically significant difference in primary bile acids (P Ͻ 0.01). The bile acid composition of the ridinilazole group more closely ap-proached that of a cohort of healthy subjects (disease free, no antibiotic exposure). To determine whether differences in bile acid levels and composition could reflect differences in BSH activity, the relative abundance of BSH genes was estimated using PICRUSt2. The predicted BSH gene abundance in vancomycin-treated subjects at EOT was more than threefold lower than in ridinilazole-treated subjects (P Ͻ 0.0001; [fig_ref] Figure 3: Bile salt hydrolase [/fig_ref]. Primary and secondary bile acids are associated with distinct families of gut bacteria. We next investigated whether the variations in bile acids could be associated with enrichment or depletion of specific groups of gut bacteria. Of note, as this analysis was focused on taxa-bile acid correlations, we included two samples from ridinilazole patients (1 each on D25 and D40) and four samples from vancomycin subjects (1 on D25 and 3 on D40) that were obtained after these subjects had begun new antibiotics as well as 10 samples that were taken at the time of suspected recurrence. Across all subjects (ridinilazole, vancomycin, and healthy subjects), significant positive correlations were detected between secondary bile acids and several families in Bacteroidales (Bacteroidaceae and Rikenellaceae) and Clostridiales (Christensenellaceae, Clostridiaceae, Lachnospiraceae, and Ruminococcaceae; Supplemental [fig_ref] Table 1: Bile acids measured in this study [/fig_ref] for BA groupings. Differences in changes across time and treatment were assessed using a linear mixed-effect model (SAS 9.4). Statistically significant outcomes were relative abundances of secondary and conjugated primary BA. BL, baseline; D10, day 10; D25, day 25; D40, day family Coriobacteriaceae [fig_ref] Figure 4: Correlations of microbiota with bile acids [/fig_ref]. These bacterial families also correlate negatively with primary bile acids, suggesting that they could contribute to the enzymatic conversion of primary to secondary bile acids. An opposite trend was found for Enterobacteriaceae (Supplemental [fig_ref] Figure 4: Correlations of microbiota with bile acids [/fig_ref] ; this family of Proteobacteria correlates positively with conjugated primary bile acids and negatively with secondary bile acids. Bifidobacteriaceae show the strongest negative correlation with conjugated bile acids, consistent with genomic data showing that many species in this family harbor BSH [bib_ref] Bile salt biotransformations by human intestinal bacteria, Ridlon [/bib_ref]. These trends largely hold for ridinilazole and vancomycin samples analyzed together (healthy controls excluded), although there are fewer significant correlations [fig_ref] Figure 4: Correlations of microbiota with bile acids [/fig_ref]. In contrast, samples from healthy subjects alone lack any significant correlations (data not shown). The number of significant correlations is further reduced when the ridinilazole and vancomycin samples are analyzed separately (Supplemental [fig_ref] Figure 5: Untargeted analysis of end-of-treatment [/fig_ref] , https://doi.org/10.6084/m9.figshare.11859252.v2). Between the two antibiotics, fewer correlations were detected for the vancomycin group. Whereas ridinilazole samples still show significant correlations involving Enterobacteriaceae (negative correlation) and several families in Clostridiales (positive correlations), these correlations are no longer detected in the vancomycin samples. Overall, this suggests that detection of correlations is primarily driven by the magnitude of OTU and bile acids variations resulting from CDI and/or antibiotic treatment. ## Untargeted analysis corroborates differences in bile acid metabolism between ridinilazole-and vancomycin-treated subjects. To determine whether there were other major metabolic differences between the ridinilazole and vancomycin groups, we more broadly characterized the subjects' fecal metabolome using untargeted LC-MS experiments. We focused this analysis on EOT, as this time point showed the greatest differences in both bile acids and predicted metagenomes. A scatterplot of the first two principal component scores (PC1 and PC2) derived from all monoisotopic LC-MS features shows nearly complete separation of ridinilazole and vancomycin samples along PC1 [fig_ref] Figure 5: Untargeted analysis of end-of-treatment [/fig_ref]. The PCA result was corroborated by a multivariate test (PERMANOVA, P Ͻ 0.001) as well as statistical tests on individual features. After FDR correction, we found 4,066 features that were present at significantly different levels in ridinilazole and vancomycin groups. More than 83% of these features were elevated in the ridinilazole group. Using our automated data annotation procedure, we putatively identified 142 significant metabolites that could be mapped to at least one functional category, i.e., KEGG pathway map, predicted by PICRUSt2. Approximately 87% of the putatively identified metabolites were elevated in the ridinilazole group, similar to the overall feature table. Pathway analysis using MetaboAnalyst [bib_ref] MetaboAnalystR: an R package for flexible and reproducible analysis of metabolomics data, Chong [/bib_ref] found four pathways that are overrepresented in the putatively identified set of metabolites detected at significantly different levels. Half of these pathways were related to bile acid metabolism, specifically taurine metabolism (P Ͻ 0.01) and primary bile acid synthesis (P Ͻ 0.05). The bile acid trends from the untargeted analysis are in good agreement with the results from targeted analysis (Supplemental , https://doi.org/ 10.6084/m9.figshare.12217949.v1). We also detected a significantly higher level of free taurine in the ridinilazole group, consistent with the increase in taurine-conjugated bile salts in the vancomycin group [fig_ref] Figure 5: Untargeted analysis of end-of-treatment [/fig_ref]. Secondary bile acids correlate with recurrence. Because of the limited number (only 1) of ridinilazole-treated subjects with confirmed CDI recurrence, a statistical analysis on the differences between treatment groups in the subset of subjects with recurrence could not be performed. To explore whether bile acid profiles correlated with recurrence regardless of the treatment, we compared samples from all subsequently recurrent (n ϭ 7 subjects) and nonrecurrent subjects (n ϭ 35 subjects) in the entire study cohort at EOT . There was no association between recurrence and any of the individual primary bile acids (data not shown). However, subjects who subsequently suffered from a recurrence of CDI had a significantly lower level of secondary bile acids. Additionally, subjects with recurrence had a significantly higher ratio of total conjugated to secondary bile acids. The concentrations of conjugated primary bile acids were lower in subjects who did not recur compared with those who did, but this difference was not statistically significant. Primary bile acid and taurine concentrations were not different. # Discussion ## Comparison of bile acid profiles between ridinilazole-and vancomycin-treated subjects. This is the first study to demonstrate in humans the relationships between CDI antibiotic treatment choice and bile acid metabolism both during therapy and after treatment cessation. We discovered that ridinilazole and vancomycin had distinct impacts on stool bile acid profiles in CDI subjects at EOT and posttherapy [fig_ref] Figure 2: Bile acid [/fig_ref]. Vancomycin treatment resulted in a significant increase of primary and conjugated bile acids, which was accompanied by a significant decrease in secondary bile acids at EOT compared with baseline. Our results on vancomycin-treated subjects with CDI resemble bile acid profiles associated with vancomycininduced dysbiosis described in previously published human studies on non-CDI subjects with inflammatory bowel disease and primary sclerosing cholangitis [bib_ref] A pilot study of fecal bile acid and microbiota profiles in inflammatory..., Vaughn [/bib_ref] , obesity [bib_ref] Effects of gut microbiota manipulation by antibiotics on host metabolism in obese..., Reijnders [/bib_ref] [bib_ref] Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin..., Vrieze [/bib_ref] , or prediabetes [bib_ref] The effects of amoxicillin and vancomycin on parameters reflecting cholesterol metabolism, Baumgartner [/bib_ref]. Similar alterations in stool bile acid profile following vancomycin administration have been observed in murine studies [bib_ref] Reduction in hepatic secondary bile acids caused by short-term antibiotic-induced dysbiosis decreases..., Kuno [/bib_ref] [bib_ref] Antibiotic-induced alterations of the gut microbiota alter secondary bile acid production and..., Theriot [/bib_ref]. In striking contrast to vancomycin, Gene abundance was estimated from taxonomic abundance data [operational taxonomic unit (OTU) table] using PICRUSt2. Dot plot shows estimated bsh abundance for ridinilazole-(RID; blue circles) and vancomycin-treated (VAN; red squares) subjects. Box plot shows medians and interquartile ranges. *P Ͻ 0.0001, statistically significant difference between treatment arms by the Wilcoxon rank-sum test. G232 ANTIBIOTIC IMPACT ON BILE ACID PROFILE ridinilazole preserved the levels of primary bile acids, conjugated bile acids, and secondary bile acids in the stool samples from CDI subjects at EOT relative to baseline. Moreover, the bile acid composition of the ridinilazole-treated subjects trended toward that of a cohort of healthy subjects 40 days after diagnosis of CDI as compared with the vancomycin group. The apparent differential effects of ridinilazole and vancomycin on bile acid homeostasis is likely due to their distinct impacts on intestinal microbiota, as metabolism of conjugated primary bile acids to secondary bile acids in the distal small intestine and colon depends on bacterial enzymes [bib_ref] Intestinal crosstalk between bile acids and microbiota and its impact on host..., Wahlström [/bib_ref]. Our previous study in this cohort showed that vancomycin treatment resulted in microbiota-wide changes at EOT with significant reductions in relative abundances of Firmicutes, Bacteroidetes, and Actinobacteria [bib_ref] Substituting whole grains for refined grains in a 6-wk randomized trial has..., Thorpe [/bib_ref]. The same study showed that ridinilazole had a markedly narrower impact on the microbiota, which could explain the improved preservation of bile acidmetabolizing capability. We here observed higher predicted BSH gene abundance [fig_ref] Figure 4: Correlations of microbiota with bile acids [/fig_ref] and higher taurine levels . The higher taurine levels are consistent with higher bile salt hydrolase activity, as this is an end product of that reaction. Finally, we observed a reduction in stool bacterial biomass after vancomycin treatment in our previous study [bib_ref] Substituting whole grains for refined grains in a 6-wk randomized trial has..., Thorpe [/bib_ref] , which also likely plays a role in decreased metabolic capacity, not just the relative abundance of organisms with bile acid-metabolizing ability. Together, these findings suggest that preserved bile acid homeostasis by ridinilazole reflects its narrow spectrum. Associations between bile acids and microbial taxa in humans. Whereas there have been a number of murine studies on the effects of antibiotic-induced dysbiosis on bile acids, only a handful have examined vancomycin, and these used vancomycin in conjunction with other broad-spectrum antibiotics, e.g., cefoperazone [bib_ref] Interactions between the gastrointestinal microbiome and Clostridium difficile, Theriot [/bib_ref]. We do not discuss these murine studies to any great extent, because there are qualitative differences in the bile acid profiles of humans and mice. Some bile acids that are quantitatively important in mice; e.g., muricholic acids are absent in humans. Furthermore, nearly all (ϳ95%) of the bile salts in mice are conjugated to taurine rather than glycine. In contrast, a majority (ϳ75%) of human bile salts are conjugated to glycine. Previous studies on bile acids and the gut microbiome in CDI subjects have largely focused on the effects of FMT. [fig_ref] Table 1: Bile acids measured in this study [/fig_ref] for bile acid abbreviations. *P Ͻ 0.05, significant correlation after false discovery rate (FDR) correction. Included in this analysis are 2 samples from ridinilazole (RID) patients [1 each on day 25 (D25) and day 40 (D40)], and 4 samples from vancomycin (VAN) subjects (1 on D25 and 3 on D40) that were obtained after these subjects had begun new antibiotics as well as 10 samples (3 RID, 7 VAN) taken at the time of suspected recurrence. 2ry., secondary bile acid; CA, cholate; CDCA, chenodeoxycholic acid; Conj. 2ry., conjugated secondary bile acid; Conj. prim., conjugated primary bile acid; DCA, deoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; Prim., primary bile acid; TCDCA, taurochenodeoxycholic acid; TLCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid. Comparing stool bile acids from subjects with recurrent CDI before and after FMT, Weingarden et al. [bib_ref] Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile..., Weingarden [/bib_ref] found increases in DCA and LCA and decreases in CA, TCA, and chenodeoxycholic acid (CDCA). Whereas the study did not explicitly draw correlations between bile acids and bacterial families, their reported trends suggest positive associations between secondary bile acids and Bacteroidaceae, Lachnospiraceae, Rikenellaceae, and Ruminococcaceae. Similar results were reported by , who used regression analysis to find that OTUs in Lachnospiraceae, Ruminococcaceae, and unclassified Clostridiales were positively and negatively correlated with secondary and primary bile acids, respectively. The study also found a negative correlation between LCA and several OTUs classified as Proteobacteria. Allegretti et al.noted that Enterobacteriaceae, a family belonging to proteobacteria, is a significant predictor of recurrent CDI (rCDI). The same study identified the ratio of DCA to GDCA as the most significant bile acid predictor for being in the rCDI cohort compared with those suffering from their first episode of CDI or healthy controls. Taken together, these previous studies consistently associate expansion of several Firmicutes families and concomitant reduction of Enterobacteriaceae post-FMT with restoration of secondary bile acids and resolution of the recurrence cycle. The above associations are also detected by our rank correlation analysis, which examined the effects of different antibiotic therapies rather than FMT. This suggests that the correlations we detected are likely driven by the distribution of bile acid-metabolizing enzymes among commensal gut bacteria and that the secondary bile acid profile differences between ridinilazole and vancomycin subjects are primarily due to their differential impact on the intestinal bacterial populations. This is further supported by the novel association we identified between bile acids and Coriobacteriaceae, which correlated positively with secondary bile acids and negatively with primary bile acids. This family includes Eggerthella lenta, which are significantly more sensitive to vancomycin (MIC 90 2 g/ mL) than to ridinilazole (MIC 90 Ͼ 512 g/mL) [bib_ref] Comparative in vitro activities of SMT19969, a new antimicrobial agent, against Clostridium..., Goldstein [/bib_ref]. Strains of E. lenta have been shown to possess enzymes that can oxidize and epimerize bile acid hydroxyl groups to produce secondary bile acids [bib_ref] Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243 T, Harris [/bib_ref]. There is weaker agreement across different studies on the positive associations between primary bile acids and specific bacterial groups. Brown et al. [bib_ref] Changes in microbiota composition, bile and fatty acid metabolism, in successful faecal..., Brown [/bib_ref] reported a positive correlation between primary bile acids and Enterococcus, Lactobacillus, and Streptococcus. Another study on FMT by found the abundance of Lactobacillus to correlate positively with conjugated primary bile acids. In our study, we found only one family, Enterobacteriaceae, which significantly correlated positively with both conjugated and unconjugated primary bile acids. In comparison, we detected many more negative correlations with both conjugated and unconjugated primary bile acids. One possible explanation for these observations is that BSH activity is more broadly distributed across different phylogenic groups, whereas the 7␣-dehydroxylating enzymes have been well documented in only a handful of species within the Lachnospiraceae (C. scindens, C hylemonae) [bib_ref] BaiCD gene cluster abundance is negatively correlated with Clostridium difficile infection, Solbach [/bib_ref] , Clostridiaceae (C. sordellii) (12), Peptostreptococcaceae (C. hiranonis) [bib_ref] Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7alpha-dehydroxylating..., Kitahara [/bib_ref] , and Coriobacteriaceae (Eggerthella lenta) [bib_ref] Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243 T, Harris [/bib_ref] families. In this light, the positive association between secondary bile acids and other families such as Bacteroidaceae could reflect their BSH activity, which is a gateway enzyme upstream of 7␣-dehydroxylation in the production of secondary bile acids. We did not detect any significant correlations between Lachnospiraceae and conjugated bile acids, suggesting that this family harbors minimal BSH activity. Similarly, very few human gut bacteria in Enterobacteriaceae harbor BSH, and thus their positive correlation with primary bile acids likely reflects an indirect association driven by the expansion of Proteobacteria in dysbiotic CDI subjects [bib_ref] Gut microbiota composition and Clostridium difficile infection in hospitalized elderly individuals: a..., Milani [/bib_ref]. Alternatively, the association between bacterial families and secondary bile acids could be explained by "crossfeeding." Well-known examples of cross-feeding by gut bacteria include the metabolism of complex plant oligosaccharides and host-derived mucins and the production of B vitamins and short-chain fatty acids. An in silico analysis of Each blue circle or red square represents, respectively, a ridinilazole (RID)-or vancomycin (VAN)-treated subject. The P value (Ͻ0.001) comparing the between-group and within-group variances of the 2 treatment groups was calculated using PERMANOVA on autoscaled LC-MS data, with Euclidean distance as the dissimilarity metric. B: dot plot shows taurine levels [reported as extracted ion chromatogram area under the curve (AUC) normalized to dry sample weight] in stool from RID-(blue circles) and VAN-treated (red squares) subjects. Box plot shows median and interquartile ranges. *P Ͻ 0.05, a statistically significant difference between treatment groups by the Wilcoxon rank-sum test after false discovery rate correction. secondary bile acid metabolism by Heinken et al.found that many gut bacteria possess genes for a part of the 7␣-dehydroxylation pathway, although only a few species encode the complete pathway. Simulations performed in this study identified pairs of bacteria that could collaboratively synthesize secondary bile acids from conjugated primary bile acids. These pairs belong to Clostridiaceae, Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae, families that our correlation analysis positively associated with secondary bile acids. The existence of collaborative pathways for bile acid metabolism spanning multiple species may explain the preservation of secondary bile acids in subjects treated with ridinilazole, which is active against C. scindens and C. hylemonae, in addition to C. difficile [bib_ref] Comparative in vitro activities of SMT19969, a new antimicrobial agent, against 162..., Goldstein [/bib_ref]. Alternatively, there may be as-yet unsequenced ridinilazole-resistant species capable of converting primary bile acids and/or their conjugated salts into secondary bile acids. Potentially, both mechanisms may be active. By comprehensively profiling representative bile acids from all four major categories, we were able to extend on previous studies to take a step toward delineating direct (enzymatic) and indirect associations between various bile acids and commensal gut bacteria. Our study is unique in the broad range of conditions under which samples were taken (healthy subjects and CDI patients, multiple time points over the course of antibiotic treatment), which enabled the discovery of a large number of significant associations, confirming in vivo metabolic capabilities that have been observed previously in vitro or in silico. We did not quantify noncanonical, unsaturated bile acids such as cholenic acid, recently shown to be depleted in patients with CDI compared with patients with non-CDI related diarrhea [bib_ref] Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections, Robinson [/bib_ref]. Although their role in CDI is unclear, it has been hypothesized that these bile acids may indicate an "extended host-microbiome dihydroxylation network." Further studies are warranted to determine whether noncanonical bile acids are associated with particular taxa that are depleted in CDI and/or rCDI and, if so, whether these bile acids rebound following treatment, depending on treatment choice. Despite one small case series that has shown reduced CDI relapse rates after administration of the secondary bile acid ursodeoxycholate compared with estimated historical control rates [bib_ref] Repurposing an old drug for a new epidemic: ursodeoxycholic acid to prevent..., Webb [/bib_ref] , preservation of bile acid-metabolizing capacity is unlikely to be the sole determinant of sustained clinical response, and other metabolitesare likely also important in restoring resistance to infection. However, restoration of normal bile acid homeostasis does appear to be a proxy marker for successful FMT. The number of subjects who tested positive for recurrence of CDI in the phase 2 clinical trial for which we had samples is relatively small (6 of 22 and 1 of 22 in the vancomycin and ridinilazole groups, respectively). Although we detected a statistically significant difference between recurrence and nonrecurrence cases, the small sample size limits the statistical power. Further studies are warranted to determine whether bile acids and/or taurine, a product of bile salt deconjugation, at EOT could indicate an individual's likelihood of having a sustained clinical response. In conclusion, ridinilazole is a promising, targeted-spectrum CDI antimicrobial that minimally disrupts commensal colonic flora and the associated bile acid metabolome. This microbiotaand metabolome-preserving property of ridinilazole may ex- . Bile acid levels at end of treatment (EOT) in confirmed recurrence vs. nonrecurrence subjects. Dot plot shows concentrations of bile acids or bile acid ratios at EOT for subjects who had (n ϭ 7 subjects) or did not have (n ϭ 35 subjects) a subsequently confirmed recurrence of C. difficile infection (CDI) during the study. See [fig_ref] Table 1: Bile acids measured in this study [/fig_ref] for bile acid groupings. Box plot shows median and interquartile ranges. Data shown are log10-transformed concentrations (mol/g dry stool weight) of primary bile acids (A), conjugated primary bile acids (B), and secondary bile acids (C) and ratio of total conjugated (conjugated primary and conjugated secondary) to secondary bile acids (D). *P Ͻ 0.05, statistically significant difference between recurrence and nonrecurrence subjects by Wilcoxon rank-sum test. plain its favorable sustained clinical response rates compared with vancomycin treatment in the phase 2 trial. # Acknowledgments We thank Janis L. Breeze, MPH, Associate Director in the BERD Center at Tufts CTSI for assistance with statistical Yanagi analyses. ## Grants This work was supported by the National Science Foundation (1337760 to K. Lee), National Cancer Institute (CA211839 to K. Lee), and Summit Therapeutics, Plc (Abingdon, UK) (sponsored research agreement to K. Lee and C. M. Thorpe). X. Qian received a fellowship from the National Center for Advancing Translational Sciences, National Institutes of Health (TL1TR002546). K. Yanagi received a fellowship from Tufts University. # Disclosures A. V. Kane has received research funds from Summit Therapeutics, Plc, and a travel grant from Summit Therapeutics, Plc. D. R. Snydman has received research funds from Merck, Pfizer, and Summit Therapeutics, Plc, and has been an advisor to Merck and Summit Therapeutics, Plc. R. J. Vickers is an employee of and holds share options in Summit Therapeutics, Plc. K. Lee has received research funds from Summit Therapeutics, Plc. C. M. Thorpe has received research funds from Merck (formerly Cubist/Optimer), Actelion, and Summit Therapeutics, Plc., has been on a Summit Therapeutics, Plc, Advisory Board, and has received a travel grant from Summit Therapeutics, Plc. # Author contributions A.K., D.R.S., R.J.V., K.L., and C.M.T. conceived and designed research; K.Y. and A.K. performed experiments; X.Q., K.Y., A.K., N.A., M.L., and K.L. analyzed data; X.Q., K.Y., A.K., K.L., and C.M.T. interpreted results of experiments; X.Q., K.Y., and K.L. prepared figures; X.Q., K.Y., K.L., and C.M.T. drafted manuscript; X.Q., K.Y., A.K., K.L., and C.M.T. edited and revised manuscript; X.Q., K.Y., A.K., D.R.S., R.J.V., K.L., and C.M.T. approved final version of manuscript. [fig] Figure 2: Bile acid (BA) composition in treated subjects and healthy controls. BA composition was calculated for ridinilazole (RID; A), vancomycin (VAN; B), and healthy subjects (C) as %total/g dry wt of stool. Corresponding total BA concentrations are shown in D-F, respectively. See [/fig] [fig] Figure 3: Bile salt hydrolase (BSH) gene abundance at end of treatment (EOT). [/fig] [fig] Figure 4: Correlations of microbiota with bile acids. A: overall bile acid-microbiota correlations for all (treated ϩ control) subjects, grouped by bacterial family. B: aile acid-microbiota family correlations in treated subjects, grouped by family. Heatmap shows Spearman's rank correlation values. Operational taxonomic units (OTUs) were excluded from the correlation analysis when detected in Ͻ5% of the samples. See [/fig] [fig] Figure 5: Untargeted analysis of end-of-treatment (EOT) samples. A: scatter plot of the first 2 principal component (PC) scores of liquid chromatography-mass spectrometry (LC-MS) features. Numbers in parentheses show %variation explained by the corresponding PC. The LC-MS data were autoscaled before principal component analysis. [/fig] [table] Table 1: Bile acids measured in this study [/table]

Adipose tissue area as a predictor for the efficacy of apatinib in platinum-resistant ovarian cancer: an exploratory imaging biomarker analysis of the AEROC trial Background: Vascular endothelial growth factor (VEGF)-targeted therapy is effective in patients with ovarian cancer. Whether adipose tissue (AT) could predict the efficacy of VEGF receptor (VEGFR) inhibitors in ovarian cancer is unknown. We aimed to evaluate the ability of distinct AT depots to predict the efficacy of apatinib, a VEGFR inhibitor, in recurrent ovarian cancers included in the AEROC trial. Methods: The AEROC was a single-arm phase 2 trial of apatinib and oral etoposide in patients with platinumresistant or platinum-refractory ovarian cancer. Apatinib was administered continuously, and oral etoposide was administered every 21 days for a maximum of six cycles. This was a post hoc study based on the AEROC trial. Areas of visceral AT (VAT), subcutaneous AT (SAT), and intermuscular AT (IMAT) were measured using computed tomography scan at baseline to assess their association with the objective response rate, progression-free survival, and overall survival. Results: Of the 35 treated patients, 31 patients with at least one post-baseline efficacy assessment by computed tomography scan were included in this study. After adjusting for apatinib exposure, high VAT (odds ratio [OR], 0.16; 95% confidence interval [CI], 0.03-0.90, P = 0.037) and SAT (OR, 0.16; 95% CI, 0.03-0.87, P = 0.034) were significantly associated with a higher objective response rate. Further, decreased risks of disease progression and death were associated with high VAT (hazard ratio [HR], 0.39; 95% CI, 0.17-0.92, P = 0.031, and HR, 0.12; 95% CI, 0.04-0.40, P < 0.001, respectively), SAT (HR, 0.35; 95% CI, 0.15-0.83, P = 0.027, and HR, 0.24; 95% CI, 0.08-0.67, P = 0.007, (Continued on next page) (Continued from previous page) respectively), and IMAT (HR, 0.20; 95% CI, 0.06-0.74, P = 0.016, and HR, 0.13; 95% CI, 0.03-0.62, P = 0.011, respectively). Conclusions: High areas of VAT, SAT, and IMAT were significantly associated with better outcomes in patients with platinum-resistant or platinum-refractory ovarian cancer who received VEGFR inhibitors. AT assessments may be valuable as patient-specific imaging biomarkers for predicting response to VEGFR inhibitors. Trial registration: ClinicalTrials.gov identifier: NCT02867956. Keywords: Ovarian cancer, Visceral adipose tissue, Subcutaneous adipose tissue, Intermuscular adipose tissue, Vascular endothelial growth factor receptor Background Ovarian cancer is the deadliest gynecologic malignancy and fifth most common cause of cancer mortality in women in developed countries. Angiogenesis plays an important role in the natural history of ovarian cancer, promoting tumor growth and progression. The vascular endothelial growth factor (VEGF) signaling pathway is the most widely studied angiogenic pathway in ovarian cancer. The addition of anti-angiogenic drugs to chemotherapy, including VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs), has shown clinical benefit in terms of progression-free survival (PFS) in patients with ovarian cancer, both in the upfront and recurrent settings. However, as not all patients benefit from anti-angiogenic therapy, identification of predictive biomarkers can help with the selection of patients responsive to this treatment. There is currently a lack of reliable and validated biomarkers for predicting the outcome of patients with ovarian cancer who receive VEGFR TKIs. Obesity represents the excess accumulation of fat in adipose tissue (AT) and is known to increase the risk for quite diverse types of cancers, including endometrial cancer, colorectal cancer, breast cancer, prostate cancer, and pancreatic cancer. It is also associated with higher risks of recurrence after treatment and death in many cancer types. The mechanisms linking excess AT and cancer development are not well understood, but obesity-associated chronic inflammation is widely accepted as an important factor in carcinogenesis. Moreover, it has been demonstrated that activated adipocytes produce multiple angiogenic factors, including VEGF, leptin, fibroblast growth factor-2 (FGF-2), tumor growth factor β (TGF-β), and placental growth factor (PGF), which stimulate neovascularization during AT expansion. Indeed, adipogenesis is closely associated with angiogenesis. Recently, several studies demonstrated that the areas of visceral AT (VAT) or subcutaneous AT (SAT) measured by computed tomography (CT) may predict the outcome of patients with metastatic colorectal cancer, renal cell cancer, and melanoma, who were treated with VEGF-targeted therapy. However, these results were controversial. Apatinib is a small molecule TKI that selectively inhibits VEGFR2. Previously, we reported the outcomes of a single-arm, phase 2 study (AEROC) on the efficacy of apatinib combined with oral etoposide in patients with platinum-resistant or platinum-refractory ovarian cancer. The combination of apatinib and oral etoposide showed promising activity in this setting. In the current study, we evaluated the areas of VAT, SAT, and intermuscular AT (IMAT) using CT images that were collected prospectively in the AEROC trial. We analyzed the associations between AT areas and patient outcomes to evaluate whether distinct AT depots could predict the efficacy of apatinib in patients with platinum-resistant ovarian cancer. # Methods ## Study design The AEROC trial was a phase 2, single-arm, prospective study in patients with platinum-resistant or platinumrefractory ovarian cancer; the design and results have been previously reported. In brief, the AEROC trial evaluated the efficacy and safety of apatinib and oral etoposide in patients with platinum-resistant or platinumrefractory ovarian cancer who had been treated with at least one line of platinum-based chemotherapy. The patients were treated with apatinib at an initial dose of 500 mg daily on a continuous basis and oral etoposide 50 mg once daily on days 1-14 of a 21-day cycle, for a maximum of six cycles. The primary endpoint was the objective response rate (ORR) using the Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1. PFS and overall survival (OS) were the two main secondary endpoints. As per protocol, dose modification, including dose interruptions and dose reductions, was performed based on the severity of toxicities according to NCI Common Terminology for Adverse Events (CTCAE), version 4.03 grading system. A maximum of two dose reductions were allowed for apatinib. The first dose reduction was from 500 mg once per day continuously to 500 mg and 250 mg taken on alternate days, and the additional dose reduction was to 250 mg once daily. If the apatinib dose was reduced, the dose could not be increased later. General guidelines for apatinib dose modification were presented in the protocol (Additional file 11: study protocol). The proportion of patients who had apatinib dose reduction and the reasons for apatinib dose reductions had been described previously. The present study was a post hoc, secondary analysis of the AEROC trial. The primary objective was to evaluate the predictive value of VAT, SAT, and IMAT for the efficacy of apatinib-based treatment. The associations between areas of AT depots and ORR, updated PFS, and updated OS were investigated. Informed consent was obtained from all patients. Ethical approval was obtained by the institutional review board of Sun Yat-sen University Cancer Centre. The procedures were in accordance with the ethical standards. # Ct analysis All patients underwent CT scans before the initiation of treatment for the purpose of efficacy assessment. CT examinations were performed on a clinical 64-slice CT scanner Discovery CT750 HD (GE Medical Systems; GE, Waukesha, USA), a Dual Source CT scanner (SOMA-TOM Force; SIEMENS, Erlangen, Germany), and iCT 256 (PHILIPS, Cleveland, USA). A single slice of each patient's baseline CT image at the third lumbar vertebra (L3) was selected and analyzed by experienced radiologists blinded to patient information. The threshold was set between − 190 and − 30 Hounsfield units (HU), which is the attenuation range of AT on CT images. The VAT area was defined as intraabdominal fat bound by the parietal peritoneum or transversalis fascia, excluding the vertebral column and paraspinal muscles, with a density between − 150 and − 50 HU. The SAT area was defined as the fat superficial to the abdominal and back muscles with attenuation ranging from − 190 to − 30 HU. The IMAT area was defined as the AT area visible between muscle groups and beneath the muscle fascia with attenuation ranging from − 190 to − 30 HU. The cross-sectional surface areas (cm 2 ) of VAT, SAT, and IMAT were calculated automatically using the semiautomated GE Reformat post-processing tool (Centri-city® Radiology RA1000; GE Medical Healthcare, Little Chalfont, UK). Representative axial CT images with respect to VAT, SAT, and IMAT are shown in. ## Statistical analyses We used optimum stratification to define the optimal cutoffs for the areas of VAT, SAT, and IMAT. Optimum stratification by SAS® macros solves the threshold value of continuous covariates with binary outcomes (SAS® macros %cutpoint) and time-to-event outcomes (SAS® macros %findcut). Therefore, the cutoffs for the areas of VAT, SAT, and IMAT to assess association with the ORR were determined using SAS® macros %cutpoint, and the cutoffs to assess association with PFS and OS were determined using SAS® macros %findcut. If more than one cutoff was suggested, the value that best distinguished patients with respect to ORR, PFS, and OS was selected. These cutoffs were then used to classify patients as having high and low VAT, SAT, and IMAT. The distributions of patient characteristics according to the areas of VAT, SAT, and IMAT were compared using the chi-square or Fisher's exact tests for categorical variables and independent t test for continuous variables. The relationships between the ORR and the areas of VAT, SAT, and IMAT were assessed using logistic regressions and adjusted for the dosage of apatinib. The Kaplan-Meier curves were used to display survival distributions, and the log-rank test was used to assess the difference between patients with high and low areas of AT depots. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) for risk of disease progression and mortality associated with high and low areas of AT depots were estimated using Cox proportional hazards models. Multivariate Cox models were adjusted for the dosage of apatinib. All statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). All tests were two-sided, and the significance level was set as 0.05. # Results ## Patients From August 10, 2016, to November 9, 2017, a total of 35 patients were enrolled in the AEROC trial. All 35 patients had baseline CT images. Four patients did not have post-baseline efficacy evaluation and were excluded from the current study. Thus, 31 patients with at least one post-baseline efficacy assessment by CT scan were included in this study. The data cutoff date used for the present analysis was February 7, 2019. The median follow-up was 13.8 months (range, 4.4-25.4 months). As of the data cutoff date, 2 patients were still receiving apatinib. Optimal cutoffs for the areas of VAT, SAT, and IMAT An area of 55.53 cm 2 was selected as the optimal cutoff for VAT associated with the ORR (Additional file 1:, PFS (Additional file 2:, and OS (Additional file 3:. The optimal cutoff for the area of SAT associated with ORR (Additional file 4:, PFS (Additional file 5: , and OS (Additional file 6:was 129.28 cm 2 , and 3.28 cm 2 was selected as the optimal cutoff for the area of IMAT associated with ORR (Additional file 7:, PFS (Additional file 8: , and OS (Additional file 9: . Patients with an area below these values were classified as having low VAT, SAT, and IMAT, respectively, and patients with an area above these values were classified as having high VAT, SAT, and IMAT, respectively. Representative axial CT images of two patients were provided (Additional file 10: : one with low VAT, low SAT, and low IMAT, and the other with high VAT, high SAT, and high IMAT. Patient characteristics according to areas of VAT, SAT, and IMAT Patient characteristics according to VAT, SAT, and IMAT depots are summarized in. No significant differences in the majority of patient characteristics were observed between patients with high or low VAT, SAT, or IMAT. However, patients with high VAT (P = 0.049) and high SAT (P = 0.034) had significantly higher apatinib exposure. Apatinib exposure was higher among patients with high IMAT than among patients with low IMAT, and the difference approached but did not reach significance (P = 0.096,. ## Association of at depots with orr In univariate logistic regression, high VAT (P = 0.015) and high SAT (P = 0.015) were significantly associated with a higher ORR. High IMAT was marginally associated with a higher ORR (P = 0.066). After adjusting for the dosage of apatinib, high VAT (odds ratio [OR], 0.16; 95% CI, 0.03-0.90, P = 0.037) and high SAT (OR, 0.16; 95% CI, 0.03-0.87, P = 0.034) were still associated with a higher ORR. However, the association between IMAT and response to apatinib was not significant when controlling for apatinib exposure. ## Association of at depots with pfs Univariate analysis demonstrated that patients with high VAT, high SAT , and high IMATwere associated with a lower risk of disease progression than those with low VAT (P = 0.035), low SAT (P = 0.028), and low IMAT (P = 0.005), respectively. When adjusting for the dosage of apatinib, high VAT was associated with a 61% (HR, 0.39; 95% CI, 0.17-0.92, P = 0.031) lower risk of disease progression compared with low VAT. High SAT and high IMAT were associated with a 65% and an 80% decreased risk of disease progression, respectively. ## Association of at depots with os Univariate analysis showed a lower risk for death among patients with high VAT, high SAT , and high IMATthan patients with low VAT, low SAT, and low IMAT, respectively. With adjustment for the dosage of apatinib, high VAT, high SAT, and high IMAT were associated with an 88% (HR, 0.12; 95% CI, 0.04-0.40, P < 0.001), a 76% (HR, 0.24; 95% CI, 0.08-0.67, P = 0.007), and an 87% (HR, 0.13; 95% CI, # Discussion This secondary analysis of the AEROC trial demonstrated an association between improved outcomes and high areas of VAT, SAT, and IMAT in patients with platinum-resistant or platinum-refractory ovarian cancer who received apatinib-based therapy. High areas of VAT and SAT predicted a better response to apatinib in this cohort. Moreover, PFS and OS were significantly superior in patients with high areas of VAT, SAT, and IMAT. Although apatinib exposure was higher among patients with high VAT, high SAT, and high IMAT, the areas of VAT, SAT, and IMAT still remained significant predictors for outcome of patients treated with apatinib after controlling for apatinib exposure. To our knowledge, this is the first time that the predictive value of AT area has been evaluated in patients with platinum-resistant ovarian cancer who received VEGFR inhibitors. There are currently only a few studies on the predictive value of AT area in patients receiving VEGF-targeted treatment. In 64 patients with metastatic renal cell carcinoma treated with VEGF-targeted therapy, a high area of VAT was independently associated with shorter time to progression (HR, 3.07; 95% CI, 1.52-6.20; P = 0.002) and OS (HR, 6.26; 95% CI, 2.29-17.08; P < 0.001). In contrast, Steffens et al.demonstrated that lower areas of VAT (HR, 3.26; 95% CI, 1.36-7.62; P = 0.006, and HR, 2.97; 95% CI, 1.36-6.47; P = 0.006, respectively) and SAT (HR, 2.66; 95% CI, 1.24-5.69; P = 0.012, and HR, 3.41; 95% CI, 1.61-7.25; P = 0.001, respectively) predicted shorter PFS and OS in patients in the same setting. The observations in the current study were in line with the work of Steffens et al. and gave further support that high areas of VAT and SAT predicted better outcomes in patients treated with VEGFR inhibitors. Despite extensive efforts, much remains to be learned about the predictive value of AT in patients receiving VEGF-targeted treatment. There is increasing evidence that AT does not simply store energy but is also an essential endocrine organ, secreting a large number of inflammatory cytokines, which is favorable for tumor development. In particular, many cytokines produced by AT show angiogenic activities. This could explain why a large amount of AT is associated with high proangiogenic factor levels and therefore resistance to VEGF-targeted therapy. However, the crosstalk between angiogenesis and adipogenesis is complex. The consequences of modulation of angiogenic activity seem to be context-dependent. For instance, inhibition of VEGF-A in wild-type mice at the initial stages of high-fat food feeding causes aggravated systemic insulin resistance, which has been linked to several types of cancer. However, the same blockade in mice with preexisting AT dysfunction had the opposite effect, with an improvement in insulin sensitivity and a decrease in inflammatory factors. These findings may be a plausible explanation for the contradictory results regarding the predictive value of AT for VEGF-targeted treatment, and to some extent support our hypothesis that anti-angiogenic therapy in patients cancer with high adipose depots, who have preexisting AT dysfunction, is more likely to result in the reduction of inflammatory cytokines, which may favor a better outcome. Until now, the optimal cutoff for specific adipose depots has not been well defined. Both the median valueand various tertileshave been used as cutoffs for AT area. Optimum stratification, which is widely used for identifying the threshold value of a continuous covariate by log-rank statistics testing, was used in this study to identify the cutoffs for different adipose depots. It should be noted that the patient population in the current study was small, and it is theoretically difficult to obtain statistically significant results. Surprisingly, we found that patients with different outcomes were well separated by the cutoffs identified by optimum stratification. This may be partly attributable to the optimum stratification method used in our study. Another plausible explanation might be that the areas of VAT, SAT, and IMAT are robust predictors of good outcomes in this patient population. However, further studies are warranted to validate these findings. The adipose organ includes numerous discrete anatomical depots. Emerging evidence indicates that not all AT depots carry equivalent risk for metabolic abnormalities. However, the contribution of different AT depots to the response to VEGF-targeted therapy is unknown. Steffens et al.identified the areas of both VAT and SAT as positive predictive biomarkers for patients with metastatic renal cell carcinoma treated with VEGF-targeted therapy. However, in the study of Ladoire et al., no significant association was observed between areas of SAT and time to progression or OS in the same setting. In the current study, we demonstrated that all three adipose depots had a positive association with the outcome of patients receiving VEGF R inhibitors. To our knowledge, this is the first analysis to investigate the predictive value of IMAT for VEGFtargeted therapy. Interestingly, we further found that the largest reduction of risk of disease progression was observed in patients with high IMAT. Nevertheless, as IMAT is a novel and less well-studied AT depot, more data are needed to validate our findings. We acknowledge that the patients in our study received oral etoposide as well which might to some extent contribute to the outcome of treatment. A growing body of evidence shows the existence of crosstalk between angiogenesis and adipogenesis, providing the theoretical underpinnings of the association between AT area and outcome of treatment with apatinib. However, we could not show the difference in outcome for patients is purely attributable to the interaction of AT depots and apatinib. # Limitations Our study has several limitations. First, the AEROC trial was prospective but the current analysis was post hoc. The AEROC trial was not originally powered to evaluate the predictive value of AT for the efficacy of apatinib treatment. In addition, we did not internally validate our findings due to the small sample size. Therefore, we are not aware of the extent to which our findings could be replicated in this setting. Second, the AEROC trial was a single-arm trial with a relatively small sample size, and selection bias may affect the generalizability of the results. # Conclusions In this secondary analysis of the AEROC trial, high areas of VAT, SAT, and IMAT were significantly associated with better outcomes in patients with platinum-resistant ovarian cancer who received VEGFR inhibitor treatment. AT assessments may be valuable as patient-specific imaging biomarkers for predicting response to VEGFR inhibitor treatment and help individualize the treatment of patients with platinum-resistant ovarian cancer. ## Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12916-020-01733-4. Additional file 1:. Best cutoffs for the areas of VAT associated with the objective response rate.showed the performance of the proposed cutoffs selected by the SAS %cutpoint macro. The cutoff 33 cm 2 achieved the highest total score. However, an area of 55.53 cm 2 was selected instead of 33 cm 2 as the optimal cutoff because it was not only significantly associated with objective response rate but also associated with progression-free survival and overall survival. VAT: visceral adipose tissue; CI, confidence interval. Additional file 2:. Plot of cutoff selection for the area of VAT associated with progression-free survival. The x-axis represents the area of VAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable as cutoffs. VAT: visceral adipose tissue. Additional file 3:. Plot of cutoff selection for the area of VAT associated with overall survival. The x-axis represents the area of VAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable as cutoffs. VAT: visceral adipose tissue. Additional file 4:. Best cutoffs for the areas of SAT associated with the objective response rate.showed the performance of the proposed cutoffs selected by the SAS %cutpoint macro. Although the area of 110 cm 2 achieved the highest total score, the area of 129.28 cm 2 was selected as the optimal cutoff because it was significantly associated with objective response rate as well as progression-free survival and overall survival. SAT: subcutaneous adipose tissue; CI: confidence interval. Additional file 5: . Plot of cutoff selection for the area of SAT associated with progression-free survival. The x-axis represents the area of SAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable as cutoffs. SAT: subcutaneous adipose tissue. Additional file 6:. Plot of cutoff selection for the area of SAT associated with overall survival. The x-axis represents the area of SAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable as cutoffs. SAT: subcutaneous adipose tissue. Additional file 7:. Best cutoffs for the areas of IMAT associated with the objective response rate.showed the performance of the proposed cutoffs selected by the SAS %cutpoint macro. The cutoff 5 cm2 had the highest total score. However, an area of 3.28 cm 2 was selected instead of 5 cm 2 as the optimal cutoff because it was not only significantly associated with objective response rate but also associated with progression-free survival and overall survival. IMAT: intermuscular adipose tissue; CI: confidence interval. Additional file 8: . Plot of cutoff selection for the area of IMAT associated with progression-free survival. The x-axis represents the area of IMAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable as cutoffs. IMAT: intermuscular adipose tissue. Additional file 9: . Plot of cutoff selection for the area of IMAT associated with overall survival. The x-axis represents the area of IMAT and the y-axis shows the Wald P value. The horizontal dotted gray line indicates significance. Points above the line have a P > 0.05, and points below the line have a P < 0.05 and are suitable for selection as cutoffs. IMAT: intermuscular adipose tissue.

Mitochondria-derived methylmalonic acid, a surrogate biomarker of mitochondrial dysfunction and oxidative stress, predicts all-cause and cardiovascular mortality in the general population A B S T R A C TBackground: Inherited methylmalonic acidemia is characterized by mitochondrial dysfunction, oxidative stress, and damage of mitochondria-rich organs in children. It is unclear whether methylmalonic acid (MMA) is related to poor prognosis in adults. The study aims to investigate the associations of MMA with all-cause and causespecific mortality in the general population. Methods: Overall, 23,437 adults from the US National Health and Nutrition Examination Survey (NHANES) were enrolled. NHANES 1999NHANES -2004NHANES and 2011NHANES -2014 were separately used as primary and validation subsets (median follow-up 13.5 and 2.8 years, respectively). Circulating MMA was measured with gas chromatography/mass spectrophotometry. Hazard ratios (HR) were estimated using weighted Cox regression models. Results: During 163,632 person-years of follow-up in NHANES 1999-2004, 3019 deaths occurred. Compared with participants with MMA <120 nmol/L, those with MMA≥250 nmol/L had increased all-cause and cardiovascular mortality in the multivariable-adjusted model [HR(95%CI), 1.62 (1.43-1.84) and 1.66 (1.22-2.27), respectively]. The association was especially significant among participants with normal cobalamin. MMA remained an independent predictor of all-cause mortality occurring whether within 5-year, 5-10 years, or beyond 10-year of follow-up (each p for trend≤0.007). That association was repeatable in NHANES 2011-2014. Moreover, baseline MMA improved reclassification for 10-year mortality in patients with cardiovascular disease (net reclassification index 0.239, integrated discrimination improvement 0.022), overmatched established cardiovascular biomarkers C-reactive protein or homocysteine. Conclusions: Circulating level of mitochondrial-derived MMA is strongly associated with elevated all-cause and cardiovascular mortality. Our results support MMA as a surrogate biomarker of mitochondrial dysfunction to predict poor prognosis in adults. The biological mechanisms under cardiovascular disease warrant further investigation. # Introduction Rapidly increasing morbidity and mortality of chronic disease remain one of the leading health challenges in the 21st century worldwide. Mitochondrial impairment and redox imbalance have been implicated in the pathogenesis of various chronic diseases, such as coronary heart disease, heart failure, dementia, and diabetes. Our previous experimental studies suggested improving mitochondrial injury and oxidative stress via genetic and pharmaceutical treatments could alleviate the severity of diabetic cardiomyopathy and myocardial infarction in mice model. Mitochondria have been considered as the key regulatory center uniquely located at the crossroads of multiple cellular processes. Targeting mitochondrial processes seem one of the most promising approaches to identify novel disease biomarker and therapeutic strategy. Methylmalonic acid (MMA), a mitochondrial intermediate metabolite of odd-chain fatty acids and several amino acids (isoleucine, valine, methionine, and threonine) catabolism, is converted into succinic acid and enters the Krebs cycle under normal conditions. The metabolism of MMA relies on healthy mitochondria. This process can be hindered by mitochondrial methylmalonyl-CoA mutase (MUT) deactivation or coenzyme active Vitamin B12 (cobalamin, Cbl) deficiency, leading to MMA accumulation. It is noteworthy that, increasing findings evidence a key role of MMA in mitochondrial dysfunction and oxidative stress both in vitro and in vivo, partly by disturbing the mitochondrial respiratory chain and inducing reactive oxygen species (ROS) generation. Inborn methylmalonic acidemia causes serious injury in mitochondria-rich organs, including cerebropathy nephropathy and cardiomyopathy in children. Blood MMA may be a favorable candidate marker of mitochondrial dysfunction and oxidative stress to predict adverse outcomes for chronic disease. Currently, circulating MMA has been used to screen genetic methylmalonic acidemia and Cbl deficiency in clinic. The prognostic value of MMA has not been reported in adults. To extend the clinical understanding of postnatal MMA increase, the study determines the association between circulating MMA and all-cause or cause-specific mortality in the general population. # Methods ## Study population All data were extracted from the National Health and Nutrition Examination Surveys (NHANES) conducted by the Center for Disease Control and Prevention of the United States. As described in our previous reports, NHANES is an ongoing, national, stratified, and multistage probability sample to investigate the health status of the non-institutionalized civilian population of all ages. In the current study,,661 participants 20 years or older in five 2-year NHANES cycles were included. We excluded individuals without eligible MMA data (n = 3192) and loss of follow-up for mortality status (n = 32). Lastly, 23, 437 people were available for analysis. The first 3 cycles NHANES 1999-2004 were combined as a subset for primary analysis (n = 13,259) and the latter two were combined to validate the relationship between MMA and mortality risk (n = 10,178). The research protocols were approved by the ethics review board of the National Center for Health Statistics. All participants provided informed consent. Information in detail is available on the website http://www.cdc.gov/nchs/nh anes/irba98.htm. ## Circulating mma measurement Blood was collected via venepuncture in mobile examination centers (MEC) according to the standardized protocols. MMA was determined in plasma and/or serum, preferentially in plasma. Prior studies confirmed MMA concentrations in serum and plasma having a common reference range and similar coefficient of variation (CV). The detection protocol has been described in detail on the website of NHANES. Due to both isomers with the same molecular weight, it had been validated that MMA could be distinguished from succinic acid through chromatographic separation. The measurement of MMA was conducted utilizing gas chromatography/mass spectrophotometry. In brief, 275 μL specimens with added internal standard solution supplemented by isotope-labeled methyl-d 3 -malonic acid (d 3 MMA) were extracted and subsequently derivatized with cyclohexanol to produce a dicyclohexyl ester. After the procedures of derivatization and separation, the effluent part from the gas chromatograph was monitored with a mass selective detector by the selected ion monitoring process. MMA level was quantitated by the peak area ratios of MMA and isotope-labeled d 3 MMA. There was a favorable linear pattern for MMA in the range of 50-2000 nmol/L. Samples with MMA concentrations >400 nmol/L were repeatedly analyzed. The total CV was 4-10% and the mean recovery rate was 96.0 ± 1.9%, as described in NHANES laboratory protocols. ## Definition of covariates General information on age, sex, race/ethnicity, family income, smoking status, physical activity, and drink was collected using standardized questionnaires during interviews. Race/ethnicity was categorized as non-Hispanic white, non-Hispanic black, Hispanic-Mexican and the other. Smoking status was classified as never, ever, and current smoking. Ever smokers were defined as participants who ever smoked at least 100 cigarettes but quitting now. Leisure physical activity during the past month was classified as vigorous, moderate, and less exercise as our previous described. Physical examinations were conducted in MEC. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared. Average systolic blood pressure (BP) or diastolic BP was estimated as the mean of eligible three readings. Laboratory examination was performed under fasting or non-fasting conditions following the standardized procedures. Samples with fasting status were defined as the time of last ate or drank anything other than water until venipuncture ≥8 h. Laboratory data on triglyceride (TG), total cholesterol (TC), High-density lipoprotein-cholesterol (HDL-C), HbA1c, insulin, alanine aminotransferase (ALT), creatinine, MMA, and Cbl were measured in all cycles, but C-reactive protein (CRP) and Hcy were just determined in NHANES 1999-2004. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). Diabetes mellitus was identified by anti-hyperglycemic therapy or HbA1c ≥ 6.5%. Hypertension was defined as antihypertensive medication, the average systolic BP ≥ 140 mmHg, or diastolic BP ≥ 90 mmHg at baseline. Cardiovascular disease (CVD) was defined as a self-reported history of coronary heart disease, heart failure, or stroke according to previous NHANES reports. Chronic obstructive pulmonary disease (COPD) was defined as emphysema or chronic bronchitis. Malignancy was self-reported. ## Abbreviations ## Aic ## Death ascertainment All participants aged 18 years or older in NHANES were linked to the National Death Index with a unique sequence number until December 31, 2015. The leading cause of death was ascertained according to the codes of International Classification of Diseases 10th Revision (ICD-10). We assessed all-cause mortality and mortality due to CVD (heart disease: I00-I09, I11, I13, I20-I51; and cerebrovascular disease: I60-I69), cancer (C00-C97), chronic lower respiratory diseases (CLRD, J40-J47), Alzheimer's disease (G30), diabetes mellitus (E10-E14), and kidney disease (N00-N07, N17-N19, N25-N27). The primary endpoint was defined as all-cause and cardiovascular mortality. # Statistical analysis Statistical analysis was conducted with Stata (version 15.0) and R (version 3.6). All estimates were weighted with masked variance of primary sampling unit, pseudo-strata and appropriate sampling weights to account for the complex sampling design unless otherwise noted. Continuous and categorical variables are expressed as weighted mean (SE) and percentage. Partial correlation analysis adjusted for age, sex, and race between the natural logarithm of MMA and cardiometabolic markers was conducted using Pearson correlation. Restricted cubic spline with age-, gender-and race-adjusted Cox regression model was used to visualize the relationship between logtransformed MMA and all-cause mortality. We found that all-cause mortality was not significantly increased when MMA<120 nmo/L. There lacked a unified standard to stratify MMA levels, and prior studies defined plasma MMA ≥250 nmol/L as one of the diagnostic criteria for functional VitB12 deficiency. Thus, participants were categorized as four groups according to MMA level: <120 nmol/L, 120-175 nmol/L, 175-250 nmol/L, and ≥250 nmol/L. Trend analysis of baseline characteristics across MMA strata was evaluated by linear regression for continuous variables and logistic regression for categorical variables after weighting. The Poisson distribution was used to estimate mortality rates and 95% CI, presented as mortality per 1000 person-years of follow-up. Crude and multivariable Cox proportional hazards regression were applied to assess the association of MMA with all-cause and causespecific mortality. Two adjusted models were adopted. Model 1 was adjusted for age (years, continuous), sex (female or male), and race/ ethnicity (non-Hispanic white, black, Hispanic-Mexican, or other). Model 2 was additionally adjusted for poverty to income ratio (<1.3, 1.3-3.5, ≥3.5, or missing), body mass index (<18. ≥30 kg/m2), smoking status (never, ever or current), alcohol consumption (no, <5, 5-30, ≥30 g/d, or missing), physical activity (inactive, moderate, or vigorous), hypertension (no/yes), fasting status (no/ yes), total cholesterol (mmol/L, continuous), HDL-C (mmol/L, continuous), CRP (mg/dL, continuous, only for NHANES 1999-2004), alanine aminotransferase (U/L, continuous), estimated glomerular filtration rate (mL/min/1.73 m 2 , continuous), CVD (no/yes), diabetes (no/yes), COPD (no/yes), cancer (no/yes), serum homocysteine (μmol/L, continuous, only for NHANES 1999-2004), cobalamin (pmol/L, continuous) and metformin use (no/yes). We further applied stratification analysis for the association between baseline MMA and all-cause mortality in subgroups by age (<65 and ≥ 65 years), sex (female and male), BMI (<30 and ≥ 30 kg/m2), current smoker (no/yes), eGFR (≥60 and < 60 mL/min per 1.73 m 2 ), Hcy (<10 and ≥ 10 μmol/L), Vit B12 (<400 and ≥ 400 pmol/L), fasting status(no/ yes), and the number of cardiovascular risk factors (≤1 and ≥ 2). The Survey-weighted Wald test was used to evaluate the potential interaction between MMA and stratification factors for mortality. Additional analysis regarding the association of MMA and all-cause mortality during 5-year bands (0-5, 5-10, and more than 10 years) was carried out to determine whether the relationship varied over time. The robustness of the association between MMA and all-cause mortality was further validated in NHANES 2011-2014 with adjustment for the aforementioned potential confounders except for CRP and Hcy. The predictive value of MMA in mortality risk was assessed in CVD patients of NHANES 1999-2004 and 2010-2014 separately. The reference model was derived from the Framingham risk score, including age, sex, Charlson comorbidity index, systolic BP, TC, and HDL-C. Goodness of Fit was assessed using the likelihood ratio test, Akaike information criterion (AIC), and Bayesian information criterion (BIC). Harrell's C-index, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) were used to assess the ability of discrimination. All tests with a 2-sided P < 0.05 were considered statistically significant unless otherwise noted. # Results ## Participant characteristics Baseline characteristics across MMA strata of the 13,259 participants in NHANES 1999-2004 were shown in. Overall, the mean age was 46.2 years and 52% were female. Participants with higher MMA levels were more likely to be older and have a higher proportion of nonblack. They were also more often taking less exercise, suffering from comorbidities, and having higher levels of cardiac risk factors, including systolic BP, CRP, TC, Hcy, and creatinine. Baseline characteristics in the validation subset NHANES 2011-2014 were comparable to NHANES 1999-2004 except for serum Hcy and CRP. Geometric mean of blood MMA at baseline was 136.5 nmol/L and their concentrations were less variable from 1999-2000 cycle to 2013-2014 cycle. There was a minor difference in MMA level between nonfasting and fasting status . ## Correlation of mma with cardiometabolic biomarkers Partial correlation coefficients adjusted for age, gender, and race were shown in. Circulating MMA was positively correlated with serum creatinine and Hcy (r = 0.25 and 0.35, respectively), as well as inversely correlated with serum Cbl (r = -0.3). Whereas, the correlation with blood pressure, BMI, glucose, HbAc1, insulin resistance index, lipid The restricted cubic spline curve shows the adjusted hazard ratios of log-transformed MMA for all-cause mortality based on Cox regression model (right Y-axis, treating MMA level = 120 nmol/L as the reference). Knots include the 5th, 27.5th, 50th, 72.5th, and 95th percentiles of the natural logarithm of MMA. The model was adjusted for sex, age, and race/ethnicity. The solid line represents point estimates, and dashed lines represent 95% CIs. The association of log MMA with mortality was J-shaped. Participants with MMA more than 120 nmol/L may have an increased risk of all-cause mortality. metabolism, CRP, and liver function was insignificant or weak. Thus, MMA might serve as a potential biomarker distinctive from traditional risk factors. Since 34.5% had laboratory blood tests under non-fasting status, the correlation analysis was further restricted to participants with fasting time ≥ 8 h. The associations remained unchanged. ## Association of mma with mortality in nhanes 1999-2004 Over a median follow-up of 13.5 (interquartile range, 11.8-15.0) years, 3019 individuals died, including 708 CVD and 628 cancer deaths. The weighted mortality was presented as a rate per 1000 person-years of follow-up, with 12.2 (95% CI, 11.6-12.8) for all-cause, 2.6 (95% CI, 2.4-2.9) for CVD, and 2.7 (95% CI, 2.5-3.0) for cancer mortality. There was a significant dose-response trend across MMA strata for all-cause and cause-specific mortalityand. The associations of MMA with all-cause and cause-specific mortality were further investigated by weighted Cox regression analysis. Both in crude and age-, sex-and race/ethnicity-adjusted models, participants with higher MMA concentration had an increased risk of all-cause mortalityand. After fully adjusting for 22 covariates, the association still complied with a dose-response pattern, with an elevated risk of all-cause mortality by 33% per each unit increase of logtransformed MMA (HR 1.33 95%CI 1.21-1.47, p < 0.001). When analyzed categorical MMA, the linear relationship remained significant in the fully adjusted model (p for trend <0.001). Compared with those with MMA<120 nmol/L, the HRs (95%CI) of all-cause mortality in participants with MMA 120-175 nmol/L, 175-250 nmol/L, and >250 nmol/L were 1.11 (0.98-1.26), 1.37 (1.18-1.60) and 1.62 (1.43-1.84). As expected, baseline MMA levels were robustly associated with higher mortality due to CVD, cancer, CLRD, Alzheimer's disease, diabetes mellitus, and nephropathy in univariate analysis. In age-, gender-and race-adjusted model, MMA level was not correlated with CLRD-and dementias-related deaths, while participants with higher MMA had a significant elevation of mortality due to CVD, cancer, diabetes mellitus, and kidney disease. After full adjustment for potential covariates, only cardiovascular mortality remained significantly increased across MMA strata, especially the heart-related mortality. Compared with participants with MMA <120 nmol/L, there was a 67% increased risk of heart-related mortality among those having MMA> 250 nmol/L (HR 1.67, 95%CI 1.19-2.34). However, the association between MMA level and cancer mortality was attenuated and became marginally significant. Restricted to healthy persons who were free from CVD and cancer at baseline, the relationship between elevated MMA levels and all-cause and cardiovascular mortality was relatively attenuated but remained significant. According to weighted Kaplan-Meier curves, even beyond 10 years of follow-up, all-cause mortality across MMA strata continued to diverge well over time. Consistently, according to prespecified Cox regression, an increase of MMA level at baseline was significantly associated with higher all-cause mortality for those events occurring within 5 years, 5-10 years or beyond 10 years of follow-up. In stratification analyses, the dose-response relationship between MMA and elevated all-cause mortality was largely consistent in subgroups by age (<65 and ≥ 65 years), sex, BMI (<30 and ≥ 30 kg/ m2), current smoker, chronic kidney disease, serum Hcy (<10 and ≥ 10 μmol/L), fasting status and the number of cardiovascular risk factors (all p ≥ 0.212 for interaction). However, a significant interaction on allcause mortality was noted between MMA and Vitamin B12 (p < 0.001 for interaction). The HRs(95%CIs) per each unit increase of logtransformed MMA for all-cause deaths were 1.25 (1.13-1.38) among participants with B12 < 400 pmol/L versus 1.69 (1.35-2.12) among participants with B12 ≥ 400 pmol/L. Variables are presented as weighted proportion (%) or mean ± SE. P for trend was estimated with linear regression for continuous variables and with logistic regression for categorical variables. *CV risk factor included current smoking, hypertension, dyslipidemia, and diabetes. ## Validation of the association in nhanes 2011-2014 The relationship between MMA and all-cause, heart-related, and cancer mortality was validated in 10,178 participants of NHANES 2011-2014. During a median follow-up of 2.8 years (29,477 personyears), 342 individuals died, with a weighted incidence rate of 9.2 per 1000 person-years. According to the Kaplan-Meier curve and multivariable Cox regression analysis, the association of MMA with mortality risk remained consistent in 2 subsets with an interval of 10-yearand . ## Incremental prognostic value of mma in cardiovascular patients Based on the above results, the prognostic value of MMA in participants with prior cardiovascular disease was investigated in NHANES 1999-2004 and 2011-2014 separately. Among 1387 patients with CVD from NHANES 1999-2004, MMA was added to the reference model and improved risk stratification of long-term mortality. The likelihood ratio test suggested an improved model fit when MMA was added to the reference model (p < 0.001). The AIC and BIC were lower in the model included MMA, compared with the reference. Regarding discrimination, adding MMA to the reference model slightly increased the C-index for the prediction of all-cause mortality. In particular, the reference model plus MMA showed the highest improvement in reclassification for 10-year mortality as assessed by the continuous NRI 0.238 (0.141-0.330) and IDI 0.022(0.012-0.032), outmatched B12, homocysteine, and CRP. The incremental prognostic value of MMA for shortterm mortality remained significant among 993 cardiovascular patients in NHANES 2011-2014. # Discussion In this large prospective study of 23,437 adults recruited from a nationally representative sample of the US, our findings demonstrated that higher levels of circulating MMA were significantly associated with increased all-cause and cardiovascular mortality, especially for those participants with normal cobalamin. Such a link remained existed independent of serum cobalamin, homocysteine, and CRP whether within 5-year or beyond 10-year follow-up. The relationship was repeatable in another subset of NHANES with an interval of 10 years. MMA showed an incremental prognostic value to predict short-and long-term mortality in cardiovascular patients based on the Framingham risk model, overmatched Hcy and CRP. Several cross-sectional studies suggested that MMA was a potential risk factor of CVD, diabetic complications, and dementias. Compared with healthy controls, patients with myocardial infarction or heart failure had significantly higher MMA concentrations. However, among 300 patients with AMI, higher MMA levels were insignificantly associated with adverse outcomes during a 4-year follow-up. A limited sample size may hinder its power of test. Our results firstly demonstrated that MMA level at baseline was significantly associated with a higher risk of long-term all-cause and cardiovascular mortality in a large prospective cohort. Notably, MMA has a more excellent predictive value than Hcy or CRP in patients with cardiovascular disease. Further validation by hospital-based cohorts is required. Higher MMA was linked to the severity of diabetic peripheral neuropathy. Serum methylmalonic acid also reflected the cognitive decline in older subjects. In our current study, we did not find a robust relationship between MMA and mortality due to diabetes and Alzheimer's disease in the multivariable-adjusted model. Lower incidence might weaken the statistical effectiveness and bias the relation towards the null hypothesis. However, all those studies still thought MMA as a marker of Cbl deficiency but not mitochondrial dysfunction. Mitochondria are considered as the critical regulatory center among multiple cellular processes. Mitochondrial dysfunction characterized by impaired bioenergetics and redox imbalance is a notable hallmark of various chronic diseases including CVD and diabetes, which significantly influence health, disease development and progression, and life span. Metabolism of MMA not only relies on healthy mitochondria and mitochondrial enzymes (MUT, CblA, and CblB), MMA accumulation also directly impacts on mitochondrial electron transport chain and redox status. Currently, mitochondrial impairment induced by MMA and MMA-related metabolites is thought to be the major cause of mitochondrial ROS generation and redox imbalance in genetic methylmalonic acidemia. A recent experimental study also demonstrated that mitochondrial dysfunction of kidney tubules was a critical pathogenic mechanism of MMA-related nephropathy in MUT knockout mice. Antioxidant therapy could partly attenuate the severity of nephropathy. Thus, biological evidence supports MMA as a potential marker of mitochondrial impairment and redox disorder. Moreover, accumulative observational studies had noted that cobalamin deficiency could only explain a small part of the increase of MMA in adults. In our findings, the moderate correlations among blood Cbl, MMA, and Hcy also supported this point. Functional cobalamin deficiency was proposed to account for the status of elevated MMA and normal B12, which had been linked with oxidative stress and assumed to be caused by the oxidation of cobalamin. However, healthy mitochondria and mitochondrial enzymes may be neglected, because the response to a large dose of Cbl treatment was largely inconsistent among subjects with functional B12 deficiency. Besides, the insufficiency of functional Cbl should cause increases in both Hcy and MMA, whereas circulating MMA was moderately correlated to Hcy here (r = 0.31). Thus, a substantial part of the increased MMA level may be imputed to mitochondrial dysfunction. Indeed, prior clinical studies supported the link between MMA and oxidative stress indicators in humans, such as oxidant risk factors, isoprostanes, oxidized cysteine, as well as decreased glutathione level and antioxidant capacity. According to the analysis of fibroblasts derived from patients and controls, methylmalonic aciduria was accompanied by a decrease in mitochondrial respiration and abnormity in mitochondria morphology. In our stratification analysis, a stronger association was identified between MMA (both continuous and categorical forms) and all-cause mortality in participants with B12 ≥ 400 pmol/L, compared with that in those with B12 < 400 pmol/L. Thus, mitochondrial dysfunction and oxidative stress, as well as poor prognosis should be cautious in adults with elevated MMA, especially in those with normal B12 simultaneously. Those biological and clinical studies as well as our results support mitochondria-derived metabolite MMA as a promising surrogate biomarker for mitochondrial dysfunction and oxidative stress. Currently, MMA mainly served as a nutritional marker for monitoring B12 deficiency in adults, but not for predicting disease severity and poor prognosis. To the best of our knowledge, this is the first large prospective cohort study to demonstrate that circulating MMA is capable of predicting long-term all-cause and cardiovascular mortality independent of B12 and Hcy. In contrast with Hcy, MMA may be an underestimated prognostic biomarker for CVD. Even though both MMA and Hcy were used to detect B12 deficiency, MMA had a more sensitive and specific performance than Hcy. However, only Hcy has been endowed with clinical implications for CVD. The moderate correlation between MMA and Hcy as well as the robust association of MMA with mortality independent of Hcy suggested that MMA as a prognostic marker could not be substituted by Hcy. Furthermore, our findings suggested MMA may improve risk stratification in cardiovascular patients outmatched Hcy. MMA-related metabolism occurred in mitochondria, while Hcy metabolized in cytoplasm. As opposed to the complicated metabolism of Hcy, MMA was not subject to folate or vitamin B6. Our findings are of particular interest because the metabolism from MMA to succinate in mitochondria is potentially restorable, and therefore is more suitable to be a potentially modifiable biomarker than Hcy. Current evidence about the toxicity of MMA in mitochondrial impairment was almost based on congenital methylmalonic acidemia. The underlying mechanisms in the setting of CVD and other chronic diseases need special clarification. There is no universally accepted definition of the reference range of MMA. Several prior studies reported different ranges of plasma MMA values in normal participants, including 10-360 nmol/L, 90-250 nmol/ L, <350 nmol/L, or <370 nmol/L. Our results indicated that participants with MMA in the normal range (175-250 nmol/L) also had a 37% increased risk of mortality compared with those with MMA <120 nmol/L. The reference range of plasma MMA may need to be redefined. Fowler B et al. summarized the cause and diagnostic approach of methylmalonic acidurias. In normal subjects, the ratio of urinary MMA on creatinine (uMMA/C) was commonly less than 10 mmol/mol. The deficiency of each segment in the MMA-related metabolic process resulted in varying degrees of methylmalonic acidurias, ranging from mild elevation 10-20 mmol/mol to severe elevation over 20,000 mmol/mol. Generally, MMA level was higher in genetic disorders than Cbl deficiency. Plasma MMA level was affected by renal impairment, while uMMA/C could limit the influence of the renal function. Thus, uMMA/C may be a more suitable biomarker reflecting MMA-related metabolism, but robust evidence is required. Quantification of downstream oxidative metabolites, such as thiols, malondialdehyde, 4-hydroxynonenal, and 8-hydroxy-2 ′ -deoxyguanosine was vulnerable to the artifactual oxidation during the procedures of storage, isolation, and purification. That limited their applicability to detect large-scale samples and merge multi-center data. Although some studies noted that MMA-related metabolites, such as 2-methylcitric acid and malonic acid, might act as more pivotal mediators than MMA itself in the pathogenesis of mitochondrial injury, the metabolic processes of MMA at least supported MMA as a surrogate marker of mitochondrial dysfunction. Our results recommended further investigation of the biological mechanisms and predictive value in chronic disease, including CVD. # Limitations and strengths This study may have some strength. The use of a nationally representative sample facilitates the generalization of our findings. The study population was divided into 2 parts, NHANES 1999-2004 and 2011-2014. We observed a striking and sustained association between MMA and mortality risk in two subsets that minimized the occasionality. Our study also has several limitations. First, MMA concentrations were analyzed using EDTA-plasma or serum specimens. Nevertheless, the potential difference may be ignorable as existing studies reported a common reference range and similar CV for MMA in serum and plasma. Second, we could not distinguish inborn and postnatal increase of MMA. However, the incidence of hereditary methylmalonic acidemia was about 1/50,000-170,000 according to data from the US and Europe, which is unlikely to alter our conclusions. Third, we could not completely rule out the possibility of residual confounding unknown. Fourth, the causality could not be concluded due to the observational study design, whereas previous experimental studies demonstrated MMA-related metabolism critically mediates mitochondrial dysfunction and oxidative stress. Fifth, fasting status could slightly increase MMA concentrations. Even though the correlations between MMA and cardiometabolic markers as well as the associations between MMA and mortality in participants with fasting or not were consistent, further study should better use fasting blood for MMA detection. # Conclusions Mitochondria-derived MMA is independently and robustly associated with increased all-cause and cardiovascular mortality in the general population, especially in participants with normal cobalamin. Such link extends beyond 10 years' follow-up. Moreover, circulating MMA improves risk stratification in patients with cardiovascular disease outmatched Hcy and CRP. Our results support MMA as a surrogate biomarker of mitochondrial dysfunction more than just monitoring cobalamin deficiency. The biological mechanisms under cardiovascular disease warrant further investigation. ## Declaration of competing interest The authors declare that there is no conflict of interest.

Validation of automated Alberta Stroke Program Early CT Score (ASPECTS) software for detection of early ischemic changes on non-contrast brain CT scans Purpose In ASPECTS, 10 brain regions are scored visually for presence of acute ischemic stroke damage. We evaluated automated ASPECTS in comparison to expert readers. Methods Consecutive, baseline non-contrast CT-scans (5-mm slice thickness) from the prospective MR CLEAN trial (n = 459, MR CLEAN Netherlands Trial Registry number: NTR1804) were evaluated. A two-observer consensus for ASPECTS regions (normal/abnormal) was used as reference standard for training and testing (0.2/0.8 division). Two other observers provided individual ASPECTS-region scores. The Automated ASPECTS software was applied. A region score specificity of ≥ 90% was used to determine the software threshold for detection of an affected region based on relative density difference between affected and contralateral region. Sensitivity, specificity, and receiver-operating characteristic curves were calculated. Additionally, we assessed intraclass correlation coefficients (ICCs) for automated ASPECTS and observers in comparison to the reference standard in the test set. Results In the training set (n = 104), with software thresholds for a specificity of ≥ 90%, we found a sensitivity of 33-49% and an area under the curve (AUC) of 0.741-0.785 for detection of an affected ASPECTS region. In the test set (n = 355), the results for the found software thresholds were 89-89% (specificity), 41-57% (sensitivity), and 0.750-0.795 (AUC). Comparison of automated ASPECTS with the reference standard resulted in an ICC of 0.526. Comparison of observers with the reference standard resulted in an ICC of 0.383-0.464. Conclusion The performance of automated ASPECTS is comparable to expert readers and could support readers in the detection of early ischemic changes. # Introduction In the treatment of acute ischemic stroke, the severity and extent of an ischemic stroke lesion could be used as one of the parameters to select eligible patients for endovascular treatment.NCCT of the brain is the most widely used modality for assessment of early focal signs of ischemic damage in stroke patients. To quantify the extent of ischemia on NCCT, the Alberta Stroke Program Early Computed Tomography Score (ASPECTS) has been introduced. In ASPECTS, 10 brain regions are dichotomously scored on the presence of early ischemic stroke signs, resulting in a range of 0 to 10, with 1 point subtracted for any evidence of early ischemic change in each defined region on the CT scan. ASPECTS scoring requires a high level of expertise to detect subtle changes on NCCT in the early phase of brain ischemia. This expertise is not available in every center where stroke patients are presented. Consequently, there is considerable interrater variability. Automated tools have been developed to counter these challenges. Siemens has developed a fully automated post-processing tool to score ASPECTS on NCCT. The performance of automated software in comparison to physicians should be tested before this software is used in clinical practice as aid for physicians. In this study, we evaluated Frontier ASPECTS software for the detection of early ischemic brain changes on NCCT scans acquired on a broad range of different CT scanners. # Materials and methods ## Study design We used image data from the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in The Netherlands (MR CLEAN, MR CLEAN Netherlands Trial Registry number NTR1804. Current Controlled Trials number, ISRCTN10888758), a prospective, consecutive study which was performed in 16 stroke centers in the Netherlands. Detailed study methods and eligibility criteria were published previously. Patients with an occlusion of the intracranial carotid artery, the M1/M2 segment of the middle cerebral artery or the A1/A2 segment of the anterior cerebral artery were included in the MR CLEAN trial (n = 500). ASPECTS or the severity and the extent of early ischemic changes were not used as exclusion criteria. The MR CLEAN study protocol was approved by the central medical ethics committee of the Erasmus MC and the research board of each participating center. All patients or their legal representatives provided written informed consent before randomization. ## Imaging data and evaluation As MR CLEAN was a multicenter trial, various CT scanner models had been used to obtain the NCCT of the brain which resulted in a heterogeneous dataset with scans from all major CT scanner manufacturers (GE Healthcare, Chicago, USA; Philips Healthcare, Amsterdam, The Netherlands; Siemens Healthineers, Erlangen, Germany; Toshiba, Tokyo, Japan). All patients with NCCT images with 5-mm slice thickness were included in the current study. All baseline NCCT scans were evaluated for ASPECTS four times by expert readers who were unaware of the treatment group assignments and final outcome. The expert readers were blinded for all clinical information, except the clinically affected cerebral hemisphere. To define a reference standard for ASPECTS, every CT scan was first rated by two expert readers from a pool of eight readers to produce a consensus score for every ASPECTS region (n = 10). In case of disagreement, a consensus score was provided by a third reader. In addition, every CT-scan was rated by two expert readers from a second pool of nine readers to produce two individual ASPECTS, hereafter named as ASPECTS of observer 1 and observer 2, respectively. ## Frontier aspects The syngo.via Frontier ASPECTS prototype software (version 2.0.1, Siemens Healthcare GmbH, Erlangen, Germany) allows analyzing NCCT scans for early ischemic changes in acute stroke in the territory of the middle cerebral arteries. A probabilistic atlas has been created based on 150 normal NCCT datasets in which ASPECTS regions (caudate nucleus (CN), internal capsule (IC), insula (INS), lentiform nucleus (LN), and 6 regions in the vascular territory of the middle cerebral artery (M1-M6)) were segmented. This human brain atlas consists of ten volumes of interest for each brain hemisphere which represent the 10 ASPECTS regions. After automatically fitting of the atlas to an NCCT brain, likelihood for belonging to a specific ASPECTS region is appointed to every voxel. The likelihood of every voxel translates to the weight of the voxel-specific HU for computing the mean HU of every ASPECTS region. To exclude cerebrospinal fluid, old infarcts, bone and calcifications, and voxels that are either too dark (below 10 HU) or too bright (above 55 HU) are excluded. The relative difference in mean HU between the individual ASPECTS region in the affected hemisphere and the contralateral hemisphere is computed and presented as a percentage HU difference. By using a predefined threshold for the relative HU difference, each ASPECTS region in the affected hemisphere is classified as affected (ischemic changes detected by the software) or not affected. The number of affected regions is used to calculate an ASPECT score. ASPECTS regionspecific threshold values are used for the classification into ischemic and non-ischemic ASPECTS regions. The default threshold values were based on initial evaluations in patients in which the automated ASPECTS was optimized with CT perfusion-based infarct core assessment as reference standard. The affected cerebral hemisphere is selected automatically by the software. If needed, the automated assessed affected hemisphere side can be adjusted to match the clinically affected cerebral hemisphere. # Statistical analysis To define the optimal threshold values for the relative HU change in the ASPECTS regions and to validate the findings, the included patients were divided in a training set (proportion of whole dataset 0.2) and a test set (proportion of whole dataset 0.8) using stratified random sampling. The datasets were stratified for affected ASPECTS regions (CN, IC, INS, LN, M1-M6) and for CT-scanner manufacturer. The training set and test set were assessed for significant differences in age, sex, NIHSS at baseline, stroke side, ischemic stroke history, prestroke modified Rankin Scale, and reference standard ASPECTS using t tests and chi-squared tests. A specificity of ≥ 90% was used in the training set to find the software threshold settings with the optimal correlation between the computed ASPECTS and the reference standard to detect ischemic changes (Online Resource). Receiver-operating characteristic curves were created to calculate the area under the curve and to assess the sensitivity and specificity of the computed ASPECTS to detect ischemic changes in the test set with the thresholds defined in the training set. Bland-Altman plots were created to evaluate for systematic differences between software and reference standard. The performance of computed ASPECTS and individual observers (1 and 2) in comparison to the reference standard as well as interobserver agreement was assessed with the intraclass correlation coefficient (ICC) using a one-way random-effects, absolute agreement, single-rater/measurement model (ICC. The strictest ICC model was used because a selection of readers out of a panel of multiple expert readers assessed the ASPECTS. For the ICC, values less than 0.5 are indicative of poor reliability, values between 0.5 and 0.75 indicate moderate reliability, values between 0.75 and 0.9 indicate good reliability, and values greater than 0.90 indicate excellent reliability. In addition, agreement per region (normal and abnormal) and agreement for trichotomized ASPECTS (0-4, 5-7, 8-10) were reported. All analyses were performed with the use of the SPSS software package, version 24.0.0.1 and R, version 3.5.1. # Results ## Patients From the MR CLEAN trial, 463 patients had a baseline NCCT with a 5-mm slice thickness. The NCCTs of 4 patients could not be processed by the automated software due to reading errors, leaving 459 (> 99%) patients available for analysis. For those 459 patients, 18 different scanners from 4 different manufacturers were used for NCCT acquisition (Online Resource,. The affected cerebral hemisphere in Frontier ASPECTS matched the clinically affected hemisphere in 86% of the patients. The remaining scans (65) were adjusted to match the clinically affected brain hemisphere. Twenty-four of the 65 wrong classifications (37%) had an ASPECTS of 7-8, and 36 (55%) had an ASPECTS of 9-10 (Online Resource,. However, after correcting for the affected hemisphere on imaging, the discriminating performance did not improve significantly (Online Resource, ; Online Resource, . Stratified random sampling resulted in allocation of 104 patients (23%) to the training set and allocation of 355 patients (77%) to the test set. Due to stratification limitations, this division was not exactly a 20%/80% division. No significant differences were found between the training set and test set. ## Training set In the training set (n = 104), with software thresholds for a specificity of ≥ 90%, we found a sensitivity of 33-49% and an area under the curve (AUC) of 0.741-0.785 for detection of an affected ASPECTS region (Online Resource, table 4). ## Validation with test set The area under the curve in the test set for assessment of ischemic changes in the central regions (caudate, insular ribbon, internal capsule, and lentiform nucleus) and the cortical regions (M1-M6) was similar to the area under the curve in the training set. With the optimal threshold value from the training set for detection of ischemic changes in the central regions (5.6%), a specificity and sensitivity of 89% and 57% were found in the test set. With the optimal threshold value from the training set for detection of ischemic changes in the cortical regions (4.7%), a specificity and sensitivity of 89% and 41% were found in the test set. Overall computed ASPECTS and reference standard agreed on region normality in 2276 regions and on the presence of ischemia in 507 regions which resulted in an overall accuracy of 78%. The accuracy of trichotomized computed ASPECTS was 60%. The ICC for the full range of ASPECTS was 0.526 (0.447-0.597). Similar results were obtained with 1 threshold for all ASPECTS regions. The Bland-Altman plot for ASPECTS difference between automated ASPECTS and reference standard showed a mean difference of 0.59 (95% CI − 3.20-4.39) (Online Resource, table 5 and . The default regionspecific thresholds resulted in a lower ICC than the optimized thresholds. No differences were found in area under the curve in evaluating ASPECTS regions in scans from different vendors (Online Resource,. ## Comparison of computed aspects to observers aspects The agreement per region of computed ASPECTS with the reference standard was similar to the region agreement of the observers with the reference standard (78% and 79% versus 84% and 77%). The agreement for trichotomized ASPECTS for the computed ASPECTS and the observers was 60% and 59% versus 60% and 57%. Reference standard ASPECTS Median (interquartile range) 8 (7-9) 7 (6-9) 0 (%) 1 (1) [formula] 1 (0) 1 (%) 0 (0) 3 (1) 2 (%) 0 (0) 3 (1) 3 (%) 1 (1) 10 (3) 4 (%) [/formula] 3 (3) 18 (5) The agreement between observers (overall agreement 86% and ICC 0.667 (95% CI 0.605-0.721)) was higher than the agreement between computed ASPECTS and the reference standard. # Discussion In this study, we evaluated the performance of Frontier ASPECTS software for the detection of early ischemic brain changes on NCCT scans. Our study showed a moderate agreement between Frontier ASPECTS and the reference standard, defined as the consensus between two expert readers. The agreement was similar to the agreement between individual readers and the reference standard, but less than interobserver agreement. Frontiers ASPECTS could aid in visual evaluation of the NCCT for the detection and quantification of early ischemic changes due to acute ischemic stroke. The Frontier ASPECTS software has specific advantages. The exclusion of bone and old infarcts before assessing the mean density of the ASPECTS regions improves the accuracy of the brain parenchyma density measurements. Old brain infarcts were found in approximately 20% of the NCCT scans and should not be used in the assessment of ASPECTS. The calculation of the mean density of the regions and the relative change in density enables adaption of the threshold to optimize the tool for specific contexts and allows the future use of the relative density values instead of the dichotomous outcomes (normal/abnormal) in prediction models. Machine learning model algorithms trained on dichotomous outcome will lack this opportunity. Current disadvantages are the reading errors in a minority of scans and the mismatch in the assessment of the affected hemisphere. However, the latter can be corrected manually, which will result in a correct assessment of the ASPECTS. Although recent evidence showed a considerably lower performance of Frontier ASPECTS, this difference could be explained by our threshold optimization and the use of software version 2.0.1 instead of 1.2.0.. A major problem in evaluation of automated software to support clinical validation is the choice of reference standard. Previous studies have used as reference standard the DWI performed within a specified time frame of the NCCT, follow up NCCT, or MRI. In studies in which follow-up NCCT was used as reference, only lesions that were present on base line and follow-up imaging were scored, or the definite final infarct core on follow-up NCCT was scored. DWI-ASPECTS as reference standard could be problematic due to the potential time delay between test and reference standard of up to 2 h which could affect the results. More important are the differences in the underlying signal changes. The NCCT scan in the early phase could be completely normal when cytotoxic edema has resulted in an abnormal DWI signal. The NCCT scan will only reveal density decrease in the next phase of vasogenic edema. This might result in low sensitivity of the algorithm in the early phase after the event. Follow-up imaging as reference standard ignores the effects of intravenous thrombolysis and endovascular thrombectomy. Minor changes on NCCT could be reversible after treatment which could result in a low specificity of the algorithm. Comparing ASPECTS with perfusion changes could produce valuable information, but it would require a different approach, as abnormalities in perfusion maps, which are apparent immediately after stroke onset, do not necessarily lead to imaging abnormalities on NCCT. However, given the relevance of ASPECTS scoring for ischemic stroke treatment and the issues with human ASPECTS scoring like moderate observer agreement, the purpose of this manuscript is to assess whether software can be used to reliably automate the ASPECTS scoring and support clinicians in assessing early ischemic changes. Therefore, comparing with DWI or perfusion imaging is beyond the aim of this paper. We therefore used consensus readings of expert observers as reference standard. The only disadvantage could be that observers are not able to detect subtle abnormalities on NCCT scans which potentially could be detected by density measurements or machine learning techniques. Therefore, we aimed at a comparison with readers as the current context of automated image analysis is the support and potential replacement of a reader in order to increase the robustness of evaluation. Independent of the approach, we found other studies reporting a similar performance. When we compare the performance of Frontiers ASPECTS with Brainomix e-ASPECTS, similar sensitivity (54% vs 44-46%) and specificity (89% vs 91-94%) was found. In addition, the performance of automated ASPECTS was better than observers. A similar study analyzing relative Hounsfield unit density per region found a sensitivity of 45% and a specificity of 93% for a HU ratio threshold of < 0.94 and an area under the curve of 0.780. A study assessing RAPID@IschemaView automated ASPECTS found a performance equal to the agreement read of expert neuroradiologists. Finally, a machine learning algorithm with DWI-ASPECTS as reference standard resulted in ICC of 0.76, a sensitivity of 66%, and a specificity of 92%. This study, similar to the current study, optimized the algorithm on a training set and tested the performance on a test set. The strength of this study includes the heterogeneity of the included patients and the use of 18 different scanner types from four major CT-scanner vendors which enables a reliable translation of the study results into clinical practice. Second, the rigorous methodology with subdivision in training and test set and the multiple metrics to assess the accuracy is a strength. Third, most studies assessing ASPECTS software performance with baseline ASPECTS as reference standard use the ASPECTS assessed by observers for both consensus for the reference standard as for individual observer analysis, which created a bias in the analysis of observer agreement. To prevent this bias in our analyses, we strictly split the expert readers panel, using independent expert readers for the reference standard consensus and for individual observer analysis. A possible limitation of this study is the use of vendorspecific software, since the software could work better for CT-scans acquired on Siemens equipment. However, the discriminating performance of the software in ASPECTS regions did not differ significantly between CT-scans acquired on Siemens CT-scanners and CT-scanners of other vendors. Secondly, in our study, we used a panel of observers to provide ASPECTS, representing the variability in observers for ASPECTS in clinical practice. Most studies do not describe the use of a panel of observers to provide ASPECTS, nor does any author describe the type of ICC they use. A practical limitation is the use of a fixed specificity of ≥ 90% in the analyses. The shape of the receiver-operating characteristic curves in this study suggests a better discriminative ability between affected and unaffected ASPECTS regions for a lower specificity, and one could want to use another threshold values resulting in a change in specificity or sensitivity, depending on their context and the type of data. This software tool should not be intended as replacement of the physician. We would recommend seeing this software tool in clinical practice as an aid for physicians. Further research is needed to compare the performance of this ASPECTS software to software of other vendors. Besides this, the role of this ASPECTS software in clinical practice needs to be established by evaluating the added value in predicting outcome compared with ASPECTS based on readers. # Conclusion In conclusion, the performance of Frontier ASPECTS is comparable to expert readers and is able to support readers in the detection of early ischemic changes in a standardized way.

Oral management of a patient with down syndrome and agammaglobulinemia: a case report Background: Down syndrome is characterized by a variety of dysmorphic features and congenital malformations, such as congenital heart disease, gastrointestinal disease, and other conditions like leukemia and autoimmune disorders. Patients with Down syndrome are highly prone to respiratory tract infections, which might be fatal to them. However, there are only few available data on patients diagnosed with Down syndrome and agammaglobulinemia. In this report, we describe a case of successful prevention of post-dental treatment complications (e.g., pneumonia and other bacterial infections) in a patient with Down syndrome and agammaglobulinemia. Case presentation: A 43-year-old man with Down syndrome, untreated agammaglobulinemia, and a history of recurrent pneumonia, was referred to our clinic for tooth mobility. To reduce the risk of post-operative infections, gammaglobulin treatment and prophylactic administration of antibiotics was scheduled before the dental procedure. Furthermore, the dental treatment, which included a filling and extractions, was conducted under general anesthesia and with the supervision of a hematologist. The dental procedures were successfully performed without any post-operative infection, and the patient is undergoing follow-up care. Conclusions: The purpose of this case report was to recommend a close liaison between physicians and dentists who may encounter a similar case, and to emphasize the importance of improving oral health of immunodeficient patients to prevent infections caused by oral microbial flora. # Background Down syndrome is characterized by a variety of dysmorphic features and congenital malformations, such as congenital heart disease, gastrointestinal disease, and other conditions like leukemia and autoimmune disorders. Patients with Down syndrome are highly prone to respiratory tract infections, which constitute the most important cause of mortality in such patients at all ages. Agammaglobulinemia is one of the most common primary immunodeficiency disorders, and is characterized by the absence of immunoglobulins. In 1952, Bruton was the first physician to describe a clinical case of absence of immunoglobulins. The diseases of primary antibody deficiency (PAD) represent a class of disorders in humans in which a defective immune system fails to produce antibodies. Patients with PAD generally have low levels of immunoglobulin (Ig) A, IgG, or IgM. PAD is associated with frequent infections of the respiratory tract, skin, sinuses, and lungs. Therefore, there is a high risk of infection during dental treatment in a patient with PAD. However, there is no report of dental treatment in patients with Down syndrome diagnosed with agammaglobulinemia. Here, we report the successful prevention of postdental treatment complications, such as pneumonia and other bacterial infections, in a 43-year-old man with Down syndrome and agammaglobulinemia, through immunoglobulin administrations and prophylactic antibiotherapy. ## Case presentation Consent for publication in this report was obtained from the patient's mother. A 43-year old male patient was referred to the Clinic for Persons with Disabilities at the Dental Hospital of Tokyo Medical and Dental University (Tokyo, Japan) with a primary complaint of tooth mobility. He had a history of Down syndrome that was diagnosed at birth. He lived alone with his mother. Family history was unremarkable. He had experienced recurrent pneumonia and chronic bronchitis since he was 34 years old. Subsequent immunological assessment revealed agammaglobulinemia and B-cell deficiency (0.47%) associated with decreased CD45 RA+ naive CD4+ T-cells (4.5% of CD4+ T-cells). He had never received gammaglobulin treatment. Oral and radiographic examinations revealed alveolar bone resorption in maxillary incisors and several decayed teeth . Marginal gingivitis was observed all around the teeth. The patient's oral hygiene was very poor with dental plaque on all surfaces of his teeth. The patient had severe mental retardation and autistic features, which included difficulty in communication. Thus, with the consent of his family, it was decided to perform a comprehensive evaluation and treatment under general anesthesia as an in-patient procedure. After consultation with a hematologist, the patient received three courses of intravenous immunoglobulin (IVIG) therapy to restore and maintain his serum IgG levels above 500 mg/dL . IVIG therapy was implemented at 4 weeks, 2 weeks, and 1 day before operation. The mandibular right first molar was restored with light-cured composite resin. The maxillary left first molar, second molar, maxillary incisors, and mandibular left incisor were extracted. After extraction, sockets were sutured to prevent post-operative infection. Suture reduced the risk of rebleeding and relieved patient discomfort. Operating table was prepared in the usual fashion. For operative field, we used 0.025% benzalkonium chloride solution and normal saline solution as usual. All procedures were carried out under standard disinfection without any additional measures. Ampicillin sodium (6 g/day) was administered every 12 h intravenously, beginning in the morning before the operation and then for 4 days after the operation, following which the patient was discharged without any infection or complication. At present, the patient is undergoing follow-up care, and the marginal gingivitis has improved. He is receiving regular IVIG treatments under the care of his local physician. # Discussion The case reported here was successfully managed through the administration of gammaglobulin and antibiotics. This report describes the management of Schedule of pre-operative intravenous immunoglobulin (IVIG) therapy. Hb = hemoglobin level (g/dL); IgA = immunoglobulin A (mg/dL); IgG = immunoglobulin G (mg/dL); IgM = immunoglobulin M (mg/dL); IVIG = intravenous immunoglobulin substitution; Plt = platelet count (10,000/μL); RBC = red blood cell count (10,000/μL); WBC = white blood cell count (/μL) agammaglobulinemia in a patient with Down syndrome during oral care procedures. Autoimmune diseases are frequently observed in patients with Down syndrome, with prevalence of immune deficiency, mild to moderate T-cell and B-cell lymphopenia with decreased naive lymphocytes, impaired mitogen-induced T-cell proliferation, reduced specific antibody responses to immunizations, and defects in neutrophil chemotaxis. These abnormalities may contribute to increased susceptibility to viral infections, hematologic malignancies, and autoimmune diseases associated with Down syndrome. For invasive dental procedures, such patients are at a high risk of severe infection and septicemia caused by the spread of oral microorganisms and their toxins through circulating blood. Clinical use of IVIG therapy has increased in the treatment of patients with PADS. IVIG therapy in patients with agammaglobulinemia reduces the risk of infection. It involves therapeutic preparations of pooled polyspecific IgG, obtained from the plasma of a large number of healthy individuals. IVIG approach had a significant and positive therapeutic impact in our patient . IVIG therapy prevents many, though not all, pulmonary complications. Though they are receiving IVIG therapy, in some patients with relatively more severe antibody deficiencies, may be in high risk of chronic bacterial infections. Then a standard course of antibiotics for acute infections stemming from surgical treatment would not be sufficient in severely immunodeficient patients and may lead to rapid relapse or recurrence of infections and further morbidity, including permanent scarring and loss of function. Experienced Panoramic radiograph, radiographic examinations revealed alveolar bone resorption in maxillary incisors and several decayed teeth clinical immunologists often prescribe a course of antimicrobials that are two to three times longer than standard recommendations. In the case described here, antibiotic prophylaxis besides the induction of IVIG treatment was effective for dental treatment of the immunodeficient patient. Owing to their susceptibility to infection, immunodeficient patients require precautions during dental treatment. Dental treatment of patients with severe combined immunodeficiency has not been previously reported in dental or medical literature. Based on our experience with this patient, we recommend that IgG, IgA, and IgM should be evaluated in patients with Down syndrome before they undergo dental procedures. Delayed diagnosis of agammaglobulinemia and other PADs might result in frequent hospitalizations owing to bacterial infections, including pneumonia, which could lead to chronic lung diseases. In the current case, initial oral examination revealed poor oral hygiene. Home care is essential for a patient's oral hygiene and dental health. However, this is difficult to achieve in patients with Down syndrome owing to the intellectual impairment and decreased manual dexterity. Since patients with Down syndrome frequently experience respiratory infections, regular oral and dental examination should be performed to reduce the risk of aspiration pneumonia; it should be remembered that as they age, people with Down syndrome are more likely to present with immune deficiency syndromes related to early senescenceand innate abnormalities in the immune response. Physicians and dentists should take exceptional precautions to detect oral pathogens in these patients, since the pathogens may result in pneumonia and other severe infection. Early diagnosis and treatment, including IVIG, is essential to improve prognosis and quality of life of patients with PADs. In addition, if possible, newborn mass screening for PADs is recommended. # Conclusions The purpose of this case report was to recommend a close liaison between physicians and dentists who may encounter a similar case, and to emphasize the importance of periodontal health in immunodeficient patients to prevent infections caused by oral microbial flora. Abbreviations IgA: Immunoglobulin A; IgG: Immunoglobulin G; IgM: Immunoglobulin M; IVIG: Intravenous immunoglobulin; PAD: Primary antibody deficiency

Internalized Sexual Stigma among Lesbian, Gay, and Bisexual Individuals in Taiwan: Its Related Factors and Association with Mental Health Problems # Introduction ## Internalized stigma Internalized stigma is a process whereby individuals endorse stereotypes about their personal characteristics, such as race and ethnicity, health status, body shape, and gender or sexual orientation. Individuals with internalized stigma may anticipate social rejection, consider stereotypes to be self-relevant, and believe that they are devalued members of society [bib_ref] The paradox of self-stigma and mental illness, Corrigan [/bib_ref] [bib_ref] A modified labeling theory approach to mental disorders e an empirical assessment, Link [/bib_ref]. Internalized stigma is prevalent among individuals with mental illnesses [bib_ref] What is the impact of mental health-related stigma on help-seeking? A systematic..., Clement [/bib_ref] , HIV-AIDS [bib_ref] Internalized stigma among people living with HIV-AIDS, Lee [/bib_ref] , obesity [bib_ref] How and why weight stigma drives the obesity 'epidemic' and harms health, Tomiyama [/bib_ref] , and drug and alcohol use [bib_ref] Review of the effects of self-stigma and perceived social stigma on the..., Hammarlund [/bib_ref] as well as among racial or ethnic [bib_ref] Health and health-related correlates of internalized racism among racial/ethnic minorities: A review..., James [/bib_ref] and sexual and gender minorities [bib_ref] A systematic review of stigma in sexual and gender minority health interventions, Layland [/bib_ref]. Studies have revealed that internalized stigma affects individuals' self-esteem, medical help-seeking behavior, and health outcomes [bib_ref] What is the impact of mental health-related stigma on help-seeking? A systematic..., Clement [/bib_ref] [bib_ref] Internalized stigma among people living with HIV-AIDS, Lee [/bib_ref] [bib_ref] How and why weight stigma drives the obesity 'epidemic' and harms health, Tomiyama [/bib_ref] [bib_ref] Review of the effects of self-stigma and perceived social stigma on the..., Hammarlund [/bib_ref] [bib_ref] Health and health-related correlates of internalized racism among racial/ethnic minorities: A review..., James [/bib_ref] [bib_ref] A systematic review of stigma in sexual and gender minority health interventions, Layland [/bib_ref] ; therefore, internalized stigma should be examined in depth and suitable interventions should be identified. ## Internalized sexual stigma in lesbian, gay, and bisexual individuals Lesbian, gay, and bisexual (LGB) individuals experience multiple types of social stigma, such as structural stigma (discrimination and stigmatization at the institutional and sociocultural levels e.g., the ban on same-sex marriage) and bullying and hate crimes derived from heterosexism [bib_ref] In defense of gay children? 'Progay' homophobia and the production of homonormativity, Bryant [/bib_ref] , and they may develop internalized sexual stigma (ISS) [bib_ref] Minority stress and mental health in gay men, Meyer [/bib_ref]. Research has revealed that ISS is a multifactorial construct [bib_ref] The development of an internalized homonegativity inventory for gay men, Mayfield [/bib_ref] [bib_ref] Measurement and correlates of internalized homophobia: A factor analytic study, Ross [/bib_ref]. According to the Measure of Internalized Sexual Stigma for Lesbians and Gay Men (MISS-LG) [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] , ISS is composed of three fundamental dimensions: identity, social discomfort, and sexuality. The identity dimension corresponds to an enduring propensity to have a negative self-attitude as a member of a sexual minority and to consider sexual stigma as part of a value system and identity ("I'd prefer to be heterosexual"; "If it were possible, I'd do anything to change my sexual orientation"); social discomfort is the fear of public identification as a lesbian or gay man in social contexts and the fear of disclosure in private and professional life (e.g., "At university (and/or at work), I pretend to be heterosexual"; "It's difficult for me to say that I'm lesbian/gay, even to someone I know"); and the sexuality dimension describes the pessimistic evaluation of the quality and duration of intimate gay or lesbian relationships and a negative conception of gay or lesbian sexual behaviors (e.g., "I don't believe in love between homosexuals"; "Gay men can only have flings/one-night stands") [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref]. Health professionals should consider all dimensions of ISS when developing prevention and intervention programs for reducing ISS in LGB individuals. ## Iss in lgb individuals during early adulthood Research has demonstrated that ISS may compromise LGB individuals' mental health [bib_ref] Internalized stigma among sexual minority adults: Insights from a social psychological perspective, Herek [/bib_ref] [bib_ref] Depression, loneliness, and sexual risk-taking among HIVnegative/unknown men who have sex with..., Su [/bib_ref] [bib_ref] Internalized homophobia, mental health, sexual behaviors, and outness of gay/bisexual men from..., Xu [/bib_ref] [bib_ref] Internalized homophobia, self-esteem, social support and depressive symptoms among sexual and gender..., Wang [/bib_ref] and social relationships [bib_ref] Sexual minority stress and same-sex relationship well-being: A meta-analysis of research prior..., Cao [/bib_ref] , increase the risk of unprotected sexual behaviors [bib_ref] Internalized homophobia, mental health, sexual behaviors, and outness of gay/bisexual men from..., Xu [/bib_ref] [bib_ref] Examining the conditions under which internalized homophobia is associated with substance use..., Puckett [/bib_ref] [bib_ref] Associations between internalized homophobia and sexual risk behaviors among young black men..., Crosby [/bib_ref] , and decrease the intention to access medical care services [bib_ref] Internalized homophobia and reduced HIV testing among men who have sex with..., Pyun [/bib_ref]. ISS may also mediate the relationship between enacted stigma and depressive symptoms in young men who have sex with men (MSM) [bib_ref] Association between enacted stigma, internalized stigma, resilience, and depressive symptoms among young..., Li [/bib_ref]. As in other Asian communities, unfavorable attitudes and unfriendly behaviors toward LGB individuals in Taiwan are deep rooted in the mindset of many people and are difficult to change [bib_ref] Changing attitudes toward homosexuality in Taiwan. Chin, Cheng [/bib_ref] [bib_ref] Associations of perceived socially unfavorable attitudes toward homosexuality and same-sex marriage with..., Ko [/bib_ref] [bib_ref] Pathways to legalizing same-sex marriage in China and Taiwan: Globalization and "Chinese..., Jeffreys [/bib_ref] [bib_ref] Impacts of public debates on legalizing the same-sex relationships on people's daily..., Lin [/bib_ref] [bib_ref] Perceived attitudes toward lesbian, gay, and bisexual (LGB) issues and mental health..., Huang [/bib_ref] [bib_ref] Relationships between traditional and cyber harassment and self-identity confusion among Taiwanese gay..., Wang [/bib_ref] [bib_ref] Suicidality among gay and bisexual men in Taiwan: Its relationships with sexuality..., Wang [/bib_ref]. Given that young adulthood is a phase of life in which individuals pursue independence and explore life possibilities [bib_ref] Emerging adulthood: A theory of development from the late teens through the..., Arnett [/bib_ref] , in this period, ISS may hinder the development of self-identity, social interaction, and intimate relationships in LGB individuals. Identifying the individual and environmental factors related to ISS in LGB individuals during early adulthood can highlight factors that should receive emphasis in efforts to better educate the broader society regarding their views on LGB individuals as well as within the context of psychotherapy in the event that an LGB individual feels the need for such therapy. ## Rationale for this study Several factors regarding ISS warrant further study. First, studies have considered ISS to be a single construct [bib_ref] Shame, internalized heterosexism, lesbian identity, and coming out to others: A comparative..., Chow [/bib_ref] [bib_ref] Prospective effects of social support on internalized homonegativity and sexual identity concealment..., Lyons [/bib_ref] [bib_ref] The Internalized Homophobia Scale for Vietnamese sexual minority women: Conceptualization, factor structure,..., Nguyen [/bib_ref] [bib_ref] HIV stigma, homophobia, sexual and gender minority community connectedness and HIV testing..., Paine [/bib_ref] [bib_ref] Trajectories of internalized heterosexism among young men who have sex with men, Puckett [/bib_ref] [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref]. However, according to the MISS-LG [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] , ISS is composed of three dimensions. Whether the factors related to ISS vary across the distinct dimensions warrants further study. Second, research has identified several sociodemographic and environmental factors that are related to ISS. For example, gender [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] , age [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref] , sexual orientation [bib_ref] HIV stigma, homophobia, sexual and gender minority community connectedness and HIV testing..., Paine [/bib_ref] [bib_ref] Trajectories of internalized heterosexism among young men who have sex with men, Puckett [/bib_ref] , social support [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref] , information on sexual minorities [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref] , and perceived social stigma [bib_ref] Shame, internalized heterosexism, lesbian identity, and coming out to others: A comparative..., Chow [/bib_ref] [bib_ref] Prospective effects of social support on internalized homonegativity and sexual identity concealment..., Lyons [/bib_ref] [bib_ref] The Internalized Homophobia Scale for Vietnamese sexual minority women: Conceptualization, factor structure,..., Nguyen [/bib_ref] [bib_ref] Trajectories of internalized heterosexism among young men who have sex with men, Puckett [/bib_ref] [bib_ref] Internalized heterosexism: Measurement, psychosocial correlates, and research directions, Szymanski [/bib_ref] are significantly associated with ISS in LGB individuals. The manner in which ISS is experienced by lesbians and gay men is considerably different [bib_ref] Internalized heterosexism: Measurement, psychosocial correlates, and research directions, Szymanski [/bib_ref] ; however, whether gender moderates the relationships between ISS and individual and environmental factors has not been examined. Third, ISS is positively associated with delayed acceptance of sexual orientation among young LGB individuals [bib_ref] Association between sexual orientation acceptance and suicidal ideation, substance use, and internalised..., Ong [/bib_ref] ; however, whether age of identification of sexual orientation is significantly associated with ISS is underdetermined. Because of the significant association between early identification of sexual orientation and bullying victimization in LGB individuals [bib_ref] Victimization of traditional and cyber bullying during childhood and their correlates among..., Wang [/bib_ref] , we hypothesized that LGB individuals who identify sexual orientation at a younger age have higher ISS compared with those who identify sexual orientation at an older age. ## Aims This cross-sectional survey study in young adult LGB individuals in Taiwan had three aims. First, we examined the factors, including demographics, sexual orientation characteristics, and perceived family support, related to ISS. Second, we examined the relationships between ISS and anxiety and depression. Third, we examined the moderating effects of gender on the associations of ISS with the related factors of anxiety and depression. We hypothesized that: (1) the levels of ISS would be different among young adult LGB individuals with different demographics, sexual orientation characteristics, and perceived family support; (2) higher ISS would be associated with higher anxiety and depression; and (3) gender would moderate the relationships between ISS and the related factors of anxiety and depression. # Methods ## Participants and procedure The Investigation on Stigma among LGB Individuals in Taiwan recruited 1000 participants (500 men and 500 women). The methods used to enroll the participants and some of the study results have been described elsewhere [bib_ref] Experience of sexual orientation microaggression among young adult lesbian, gay, and bisexual..., Tsai [/bib_ref] [bib_ref] Measure of Internalized Sexual Stigma for Lesbians and Gay Men (MISS-LG) in..., Yen [/bib_ref]. In brief, we posted an advertisement on social media sites frequently used by Taiwanese LGB individuals, such as Facebook, Twitter, and the LINE messaging app, and on the Bulletin Board System and the home pages of three health promotion centers for LGB individuals between August 2018 and July 2020. The inclusion criteria were individuals aged between 20 and 30 years who identified as homosexual or bisexual and lived in Taiwan. Those who met the inclusion criteria were invited to complete the study questionnaires individually in a study room. Those who had any condition that might interfere with their understanding of the study's purpose or how to complete the questionnaire, such as impaired intellect or alcohol and substance use, were excluded from the study. Informed consent was obtained from all participants prior to assessment. Participants completed the research questionnaires in the study rooms of the psychiatry research department affiliated to a university hospital in person and being assured that their responses to the research questionnaire would be confidential. This study provided 500 new Taiwan dollars (about 18 US dollars) for every participant as a reward. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-F(II)-20180018). ## Measures ## Iss We used the 17-item Chinese version [bib_ref] Measure of Internalized Sexual Stigma for Lesbians and Gay Men (MISS-LG) in..., Yen [/bib_ref] of the MISS-LG [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] to assess the three ISS dimensions of social discomfort, sexuality, and identity in LGB individuals. The items were rated on a 5-point Likert scale from 1 (strongly disagree) to 5 (strongly agree). Some of the sample items for each dimension have been described in the introduction of this paper. The MISS-LG has two versions with the same factor structure, one for women and the other for men. A higher total dimension score indicates a higher level of ISS. According to research, the MISS-LG has satisfactory psychometric properties [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref]. The results of Rasch and confirmatory factor analyses verified that the traditional Chinese version of the MISS-LG (TC-MISS-LG) has the same three-factor structure for the different genders used among young adult LGB individuals in Taiwan [bib_ref] Measure of Internalized Sexual Stigma for Lesbians and Gay Men (MISS-LG) in..., Yen [/bib_ref]. The TC-MISS-LG scores were significantly correlated with perceived social stigma toward sexual minorities, supporting its concurrent validity; the McDonald's omega of the three TC-MISS-LG dimensions ranged from 0.67 to 0.90, supporting its acceptable-to-excellent internal consistency [bib_ref] Measure of Internalized Sexual Stigma for Lesbians and Gay Men (MISS-LG) in..., Yen [/bib_ref]. ## Perceived family support We used the 5-item traditional Chinese version [bib_ref] A Preliminary Study of Family Apgar Index, Chen [/bib_ref] of the Family APGAR Index [bib_ref] The family APGAR: A proposal for a family function test and its..., Smilkstein [/bib_ref] to measure the five components of family support: adaptability, partnership, growth, affection, and resolve. The items were rated on a 4-point Likert scale from 1 (never) to 4 (always). A higher total score indicated a higher level of perceived family support. The traditional Chinese version of the Family APGAR Index had acceptable discriminatory validity for social adaptability [bib_ref] A Preliminary Study of Family Apgar Index, Chen [/bib_ref] and congruent validity, exhibiting a significant correlation with the state of general health [bib_ref] A preliminary study of family Apgar index in the Chinese, Chau [/bib_ref]. Cronbach's α in the present study was 0.86. ## Demographic and sexual orientation factors We collected information on the participants' gender, age, education level (high school or below vs. college or above), sexual orientation (homosexual or bisexual), and age of identification of sexual orientation. ## Anxiety We used the 20-item traditional Chinese version [bib_ref] A study of the revised State-trait Anxiety Inventory, Chung [/bib_ref] of the self-administered State-Trait Anxiety Inventory-State subscale(TC-STAI-S) to assess participants' current severity of anxiety. The items were rated on a 4-point Likert scale from 1 (not at all) to 4 (very much so). A higher total TC-STAI-S score indicated more severe anxiety. The traditional Chinese version of the TC-STAI-S had acceptable test-retest reliability (Pearson's r = 0.76), internal reliability (Cronbach's α = 0.91), criterion validity (correlation with the Hamilton Anxiety Rating Scale: r = 0.69), and construct validity [bib_ref] Evaluation of psychometric properties of the Chinese Mandarin version State-Trait Anxiety Inventory..., Ma [/bib_ref]. Cronbach's α for the TC-STAI-S in the present study was 0.89. The score of 40 is commonly used to define probable clinical levels of anxiety [bib_ref] Prevalence of anxiety in patients with an implantable cardioverter defibrillator: Measurement equivalence..., Emons [/bib_ref]. ## Depression We used the 20-item traditional Chinese version [bib_ref] Depression in Taiwan: Epidemiological survey utilizing CES-D, Chien [/bib_ref] of the self-administered Center for Epidemiological Studies Depression Scale [bib_ref] A self-report depression scale for research in the general population, Radloff [/bib_ref] (TC-CES-D) to assess the frequency of depressive symptoms among participants in the month preceding the study. The items were graded on a 4-point scale from 0 (rarely or none of the time) to 3 (most or all of the time). A higher total TC-CES-D score indicated more severe depression. The TC-CES-D had good internal consistency (Cronbach's α = 0.90), 1-week test-retest reliability (intraclass correlation reliability = 0.93), congruent validity (area under the receiver operative characteristic curves for major depressive disorder = 0.88-0.90) [bib_ref] Using the CES-D in a two-phase survey for depressive disorders among nonreferred..., Yang [/bib_ref] , and construct validity [bib_ref] Factor structure of the Center for Epidemiologic Studies Depression Scale in Taiwanese..., Cheng [/bib_ref]. Cronbach's α for the TC-CES-D in the present study was 0.93. The score of 16 is commonly used for screening major depressive disorder in the general population [bib_ref] Screening for depression in the general population with the Center for Epidemiologic..., Vilagut [/bib_ref]. # Statistical analysis Data analyses were conducted using IBM SPSS software (version 20.0; IBM, Armonk, NY, USA). Participants' ISS, individual factors (demographic and sexual orientation characteristics), perceived family support, mood problems (anxiety and depression) were analyzed using descriptive statistical methods (mean, standard deviation [SD] and percentage). We first examined the skewness and kurtosis of the continuous variables and found that all the absolute values were less than 2, indicating normal distributions according to Kim [bib_ref] Statistical notes for clinical researchers: Assessing normal distribution (2) using skewness and..., Kim [/bib_ref]. The associations between individual factors and perceived family support (independent variables) and the three ISS dimensions (dependent variables) were examined using multivariate linear regression analysis. Because of multiple comparisons, the significance of the p value was adjusted to 0.017 (0.05/3) using Bonferroni correction. The associations between ISS (independent variable) and anxiety and depression (dependent variables) were also examined using multivariate linear regression analysis, which was controlled for demographics (covariates). Because of multiple comparisons, the significance of the p value was adjusted to 0.025 (0.05/2) using Bonferroni correction. The moderating effects of gender on the associations of ISS with the related factors and with anxiety and depression were examined according to the method of Baron and Kenny [bib_ref] The moderator-mediator variables distinction in social psychological research: Conceptual, strategic, and statistical..., Baron [/bib_ref]. [fig_ref] Table 1: Participants' characteristics [/fig_ref] summarizes the data on the three ISS dimensions, demographics, sexual orientation factors, perceived family support, anxiety, and depression of the 1000 participants. The mean (SD) age of participants was 24.6 years (3.0 years); nearly 90% had a college degree or above; 57% identified as homosexual; and the mean (SD) age at which they first identified their sexual orientation was 14.5 (3.9) years. The mean total scores (SD) for the dimensions of social discomfort, sexuality, and identity, based on the MISS-LG, were 16.6 (6.0), 8.9 (3.3), and 9.9 (4.2), respectively. After transforming, the mean score for each item on the dimension of social discomfort (2.4) was the highest, followed by the scores for identity (2.0) and sexuality (1.8). The mean score (SD) for the severity of anxiety was 40.8 (12.7) and slightly higher than the cutoff 40 that was commonly used for screening anxiety disorders [bib_ref] Prevalence of anxiety in patients with an implantable cardioverter defibrillator: Measurement equivalence..., Emons [/bib_ref]. The mean score (SD) for depression was 18.8 (11.2) and higher than the cutoff 16 that was commonly used for screening major depressive disorders [bib_ref] Screening for depression in the general population with the Center for Epidemiologic..., Vilagut [/bib_ref]. [fig_ref] Table 2: Factors related to internalized sexual stigma [/fig_ref] presents the results of the multivariate linear regression analysis of the associations between demographics, sexual orientation factors, and perceived family support and the three ISS dimensions. The condition index was 29.981, indicating no problem of collinearity. The results indicated that male participants had higher ISS in all three dimensions than female participants. Participants identifying as homosexual had lower ISS in all three dimensions than those identifying as bisexual. An older age of identification of sexual orientation was identified as being significantly associated with higher ISS in the dimensions of social discomfort and sexuality. Lower perceived family support was significantly associated with higher ISS in the dimension of social discomfort. # Results ## Social discomfort Sexuality Identity presents the results of the multivariate linear regression analysis of gender's moderating effects on the association between the three ISS dimensions and related factors. The results indicated that the interaction between gender and sexual orientation was significantly associated with the identity dimension of ISS, indicating that gender moderated the association between this dimension and sexual orientation. Further examination revealed that bisexual men had higher identity-related ISS than gay men (B = −2.321, SE = 0.470, p < 0.001), whereas no difference in identity-related ISS was observed between bisexual and lesbian women (B = −0.415, SE = 0.366, p = 0.257). . Moderating effects of gender on the associations of internalized sexual stigma with related factors: Multivariate linear regression analysis. ## B (se) b (se) b (se) ## Social discomfort Sexuality Identity provides the results of the multivariate linear regression analysis of the associations between ISS and anxiety and depression. The condition index was 29.929, indicating no problem of collinearity. Higher ISS in the dimension of social discomfort was significantly associated with higher anxiety and depression, higher ISS in the dimension of sexuality was significantly associated with higher anxiety, and higher ISS in the dimension of identity was significantly associated with higher depression. provides the results of the multivariate linear regression analysis of gender's moderating effects on the associations between ISS and anxiety and depression. The results indicated that the interaction between gender and the social discomfort dimension of ISS was significantly associated with anxiety, indicating that gender moderated the association between social discomfort and anxiety. Further examination revealed that social discomfort was significantly associated with anxiety in lesbian and bisexual women (B = 0.708, SE = 0.127, p < 0.001), but this was not the case for gay and bisexual men (B = 0.152, SE = 0.133, p = 0.252). ## B (se) b (se) b (se) # Discussion The present study demonstrated that gender, sexual orientation, age of identification of sexual orientation, and perceived family support were significantly associated with all or some dimensions of ISS in LGB individuals. Various dimensions of ISS had different relationships with anxiety and depression. Gender had a moderating effect on the association between the identity dimension of ISS and sexual orientation as well as the on association between the social discomfort dimension of ISS and anxiety. ## Factors related to iss The MISS-LG has two versions, one for men and the other for women, with the same three first-order factors, the same number of items, and some different item contents related to gender [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref]. It provides a good basis for comparing ISS-related gender differences. In line with the results of previous studies [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] [bib_ref] Binge drinking among gay, and lesbian youths: The role of internalized homophobia,..., Baiocco [/bib_ref] [bib_ref] Adaptation to sexual orientation stigma: A comparison of bisexual and lesbian/gay adults, Balsam [/bib_ref] [bib_ref] Sexual orientation identity and romantic relationship quality in same-sex couples, Mohr [/bib_ref] , the results of the current study revealed that gay and bisexual men reported higher ISS in all three dimensions than lesbian and bisexual women. Gay and bisexual men endure greater pressure to conform to a heteronormative gender role and are more often condemned by their heterosexual peers [bib_ref] Measure of internalized sexual stigma for lesbians and gay men: A new..., Lingiardi [/bib_ref] [bib_ref] Disclosure of sexual orientation, victimization, and mental health among lesbian, gay, and..., D&apos;augelli [/bib_ref] [bib_ref] Hate crimes and stigma-related experiences among sexual minority adults in the United..., Herek [/bib_ref]. Gay and bisexual men may experience more public stigma based on heterosexualism and thus comprehensively internalize sexual stigma more than do lesbian and bisexual women. The present study revealed that bisexual men and women had higher ISS in the dimensions of social discomfort and sexuality than gay men and lesbians. The results are congruent with those of previous studies [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref] [bib_ref] Sexual identity, mental health, HIV risk behaviors, and internalized homophobia among black..., Amola [/bib_ref] [bib_ref] Masculinity, muscularity, and HIV sexual risk among gay and bisexual men of..., Brennan [/bib_ref] [bib_ref] Correlates of internalized homonegativity among black men who have sex with men, Quinn [/bib_ref]. A 1-year longitudinal study also demonstrated that bisexual youth experienced ISS more than did gay or lesbian youth [bib_ref] Gay-related stress and emotional distress among gay, lesbian, and bisexual youths: A..., Rosario [/bib_ref]. The present study further revealed an association between the ISS dimension of identity and the sexual orientation of bisexuality in men, but not in women. Research has demonstrated that bisexual individuals experience prejudice, for example sexual orientation instability and sexual irresponsibility, from both heterosexuals and lesbians and gay men [bib_ref] Metaperceptions of others' attitudes toward bisexual men and women among a nationally..., Beach [/bib_ref] [bib_ref] Navigating the borderlands: The roles of minority stressors, bicultural self-efficacy, and cognitive..., Brewster [/bib_ref] [bib_ref] Attitudes toward bisexual men and women among a nationally representative probability sample..., Dodge [/bib_ref] , which may increase the risk of developing ISS as part of a value system and in relation to identity [bib_ref] Bisexual men's experiences with discrimination, internalized binegativity, and identity affirmation: Differences by..., Sarno [/bib_ref]. The result indicated that the effect of social prejudice on internalizing stigma toward the sexual orientation of bisexuality might be stronger in men than in women. A previous study in the United States identified that peer support, but not family support, was associated with low ISS among young MSM aged between 16 and 20 years [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref] ; the authors hypothesized that at this stage of development, peer support plays a larger role than family support in determining the experience of ISS [bib_ref] Factors predicting internalized stigma among men who have sex with men living..., Xu [/bib_ref]. However, the present study discovered that lower perceived family support was significantly associated with higher ISS in the dimension of social discomfort among young adult LGB individuals in Taiwan. Most young adults in Taiwan maintain a close relationship with their family and are concerned about the expectations and attitudes of their elders toward them. People in Taiwan are deeply influenced by Confucianism; adults who are unmarried and have no offspring are considered to have failed in observing filial piety [bib_ref] Filial piety and psychological wellbeing in well older Chinese, Cheng [/bib_ref] [bib_ref] Stressors based on sexual orientation and mental health among lesbian, gay, and..., Zheng [/bib_ref] [bib_ref] Pioneer of the health and well-being of sexual minorities in Asia, Hsu [/bib_ref]. Young adult LGB individuals may internalize their failure to meet their family obligations and develop ISS, especially with regard to the fear of disclosure in family life. The present study determined that a younger age of identifying sexual orientation was associated with higher ISS in the dimensions of social discomfort and sexuality. Because early adolescence is a stage for exploring and developing self-identification, LGB individuals who identify their sexual orientation at this stage may lack the ability to reject social stigma toward sexual minorities. They may also lack the LGB approbation that may enable them to develop positive self-identification regarding sexual orientation. These developmental perspectives may contribute to ISS development among LGB individuals with early identification of sexual orientation. ## Associations of iss with anxiety and depression The present study revealed that higher ISS in the social discomfort dimension was significantly associated with higher anxiety and depression. The relationship between ISS and mood problems might be bidirectional. According to Erikson, young adults are eager to develop intimacy with others. Young adult LGB individuals who experience social discomfort-related ISS may have difficulties in developing intimate relationships with others, and the risk of anxiety and depression may increase. Moreover, establishing sexual orientation and initiating relationships are key developmental tasks during adolescence. The ISS dimensions of sexuality and identity may be formed in early adolescence and may interfere with the accomplishment of developmental tasks at the end of adolescence; the adverse impact may persist into early adulthood and compromise emotional regulation. Alternatively, mood problems might increase LGB individuals' sensitivity to the existence of sexual stigma and might increase the severity of ISS. The present study also revealed that the social discomfort dimension of ISS was significantly associated with anxiety in lesbian and bisexual women, but not in gay and bisexual men. Studies have established that women are more likely than men to have anxiety disorders [bib_ref] Anxiety disorders: Sex differences in prevalence, degree, and background, but gender-neutral treatment, Bekker [/bib_ref] [bib_ref] Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity..., Kessler [/bib_ref] [bib_ref] Cross-national associations between gender and mental disorders in the World Health Organization..., Seedat [/bib_ref] ; however, the present study could not determine the reasons for the moderating effect of gender. Further research is required to examine this factor. ## Implications The findings of the present study highlight the value of developing strategies for the prevention of ISS in LGB individuals. While acceptance of LGB individuals has substantially improved over the past several decades, total acceptance is far from complete due to the continued prevalence of generally unwarranted negative familial and cultural stereotypes and thus further efforts to modify outdated beliefs with regard to LGB individuals are warranted. Constitutions, laws and anti-discrimination policies at the national level that include protection from discrimination on the grounds of sexual orientation is of fundamental importance. Broadening understanding of LGB culture and awareness of prejudices toward LGB individuals in educational settings, workplaces, and family environments are also the necessary steps to help reduce sexuality-related stigma. Public health strategies addressing attitudes to sexual orientation and promoting the changes of attitudes toward sexual minority among the general population may contribute to diverse affirmative cultural scripts regarding LGB individuals' lives [bib_ref] Queer narratives and minority stress: Stories from lesbian, gay and bisexual individuals..., Synnes [/bib_ref]. Although several studies have developed the programs for reducing ISS among gay and bisexual men [bib_ref] Results of a pilot study to ameliorate psychological and behavioral outcomes of..., Smith [/bib_ref] [bib_ref] A computer-based intervention to reduce internalized heterosexism in men, Lin [/bib_ref] [bib_ref] Reducing internalized homonegativity: Refinement and replication of an online intervention for gay..., Israel [/bib_ref] , there is no study directly examining the effects of interventions addressing familial and cultural stigma on ISS among LGB individuals. Meanwhile, given the effect of gender on the associations between ISS and sexual orientation and anxiety identified in this study, intervention programs should be gender-specific. # Limitations Some limitations of this study should be considered when interpreting the results of this study. First, the temporal relationships among ISS, family support, and mood problems could not be determined in this cross-sectional study. Further prospective studies are needed to examine the temporal relationships among these variables; for example, whether ISS can predict the development or change of mood problems warrants being examined in prospective studies. Second, the present study recruited a group of LGB individuals aged between 20 and 30 years. Therefore, further study is needed to examine whether the results of this study can be generalized to the populations of other age ranges. Third, we collected the data based on the participants' self-report; there might be single-rater bias. Fourth, the options for the question inquiring participants' gender identity contained man and woman only but no other gender identities such as transgender, gender nonbinary, and genderqueer. # Conclusions Gender, sexual orientation, age of identification of sexual orientation, and perceived family support were related to the ISS dimensions in young adult LGB individuals. ISS was significantly associated with anxiety and depression. Further efforts to modify the public's prejudice with regard to LGB individuals are warranted. Family education and cultural change aiming to reduce public stigma against LGB individuals are needed to mitigate ISS among LGB individuals. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data will be available upon reasonable request to the corresponding authors. ## Conflicts of interest: The authors declare no conflict of interest. [fig] 5: Moderating effects of gender on the associations of internalized sexual stigma with anxiety and depression: Multivariate linear regression analysis. on the MISS-LG 0.646 (0.120) *** 0.417 (0.110) *** Sexuality on the MISS-LG 0.333 (0.315) 0.241 (0.173) Identity on the MISS-LG 0.299 (0.123) * 0.076 (0.163) Gender x Social discomfort on the MISS-LG −0.414 (0.163) * −0.241 (0.155) Gender x Sexuality on the MISS-LG 0.265 (0.383) Gender x Identity on the MISS-LG 0.403 (0.218) MISS-LG: Measure of Internalized Sexual Stigma for Lesbians and Gay Men. * p < 0.05; *** p < 0.001. Bold:Indicates significance using Bonferroni correction, which adjusted the p value to 0.025. [/fig] [fig] Author: Contributions: Conceptualization, C.-F.Y.; Methodology, J.-I.L. and C.-F.Y.; Validation, Y.-P.C., C.-S.T. and C.-F.Y.; Formal Analysis, J.-I.L. and C.-F.Y.; Investigation, C.-F.Y.; Resources, C.-F.Y. and C.-S.T.; Data Curation, C.-F.Y.; Writing-Original Draft Preparation, J.-I.L., C.-S.T. and C.-F.Y.; Writing-Review & Editing, Y.-P.C.; Visualization, C.-F.Y.; Supervision, C.-F.Y.; Project Administration, C.-F.Y.; Funding Acquisition, C.-F.Y. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a research grant from the Ministry of Science and Technology, Taiwan (107-2314-B-037-102-MY3) and Kaohsiung Medical University Hospital (grants KMUH107-7R69, KMUH108-8R65 and KMUH109-9R77). Institutional Review Board Statement: The institutional review boards of Kaohsiung Medical University Hospital (approval number: KMUHIRB-F(II)-20180018; date of approval: 27 March 2018) approved this study. [/fig] [table] Table 1: Participants' characteristics (N = 1000). MISS-LG: Measure of Internalized Sexual Stigma for Lesbians and Gay Men. [/table] [table] Table 2: Factors related to internalized sexual stigma: Multivariate linear regression analysis. [/table]

Remote cognitive assessment in severe mental illness: a scoping review Many individuals living with severe mental illness, such as schizophrenia, present cognitive deficits and reasoning biases negatively impacting clinical and functional trajectories. Remote cognitive assessment presents many opportunities for advancing research and treatment but has yet to be widely used in psychiatric populations. We conducted a scoping review of remote cognitive assessment in severe mental illness to provide an overview of available measures and guide best practices. Overall, 34 studies (n = 20,813 clinical participants) were reviewed and remote measures, psychometrics, facilitators, barriers, and future directions were synthesized using a logic model. We identified 82 measures assessing cognition in severe mental illness across 11 cognitive domains and four device platforms. Remote measures were generally comparable to traditional versions, though psychometric properties were infrequently reported. Facilitators included standardized procedures and wider recruitment, whereas barriers included imprecise measure adaptations, technology inaccessibility, low patient engagement, and poor digital literacy. Our review identified several remote cognitive measures in psychiatry across all cognitive domains. However, there is a need for more rigorous validation of these measures and consideration of potentially influential factors, such as sex and gender. We provide recommendations for conducting remote cognitive assessment in psychiatry and fostering high-quality research using digital technologies.Schizophrenia (2022) 8:14 ; https://doi. # Introduction Cognitive impairment is a core feature of psychiatric illness, particularly schizophrenia and related disorders [bib_ref] Schizophrenia is a cognitive illness: time for a change in focus, Kahn [/bib_ref] [bib_ref] Cognitive impairment in bipolar disorder: treatment and prevention strategies, Solé [/bib_ref]. Robust cognitive deficits are observed in several cognitive domains in schizophrenia, including memory, attention, and executive function [bib_ref] Neurocognitive impairment in deficit and non-deficit schizophrenia: a meta-analysis, Bora [/bib_ref] [bib_ref] Cognitive impairment in depression: a systematic review and meta-analysis, Rock [/bib_ref] [bib_ref] Cognitive impairment in bipolar disorder and schizophrenia: a systematic review, Vöhringer [/bib_ref]. Less wellknown cognitive symptoms in schizophrenia are cognitive biases, which are errors in judgment or interpretation that affect decisionmaking (e.g., jumping to conclusions, confirmation bias) and contribute to symptoms [bib_ref] Efficacy of psychological interventions targeting cognitive biases in schizophrenia: a systematic review..., Sauvé [/bib_ref] [bib_ref] cognitive biases and assessment of certainty: a neurocognitive model of delusions, Broyd [/bib_ref] [bib_ref] The combined cognitive bias hypothesis in depression, Everaert [/bib_ref]. Both traditional cognitive impairments and elevated cognitive biases are rooted in neurobiology 9,10 and affect many diagnosed with mental illness [bib_ref] Meta-analysis of cognitive impairment in first-episode bipolar disorder: comparison with first-episode schizophrenia..., Bora [/bib_ref] [bib_ref] Cognitive impairment in psychotic illness: prevalence, profile of impairment, developmental course, and..., Mccleery [/bib_ref] [bib_ref] Comparing cognitive clusters across first-and multiple-episode of psychosis, Sauve [/bib_ref] , negatively impacting clinical and functional trajectories [bib_ref] Efficacy of psychological interventions targeting cognitive biases in schizophrenia: a systematic review..., Sauvé [/bib_ref] [bib_ref] Neurocognition: clinical and functional outcomes in schizophrenia, Lepage [/bib_ref]. Cognitive assessments are essential in guiding treatment planning and, thus, proper measurement of both cognitive capacity and cognitive biases is fundamental to improve overall patient cognitive health and outcomes. Remote cognitive assessments outside the clinic or laboratory have become a necessity in the context of the COVID-19 pandemic, which has hindered mental health initiatives in both research and clinical settings worldwide [bib_ref] A projection for psychiatry in the post-COVID-19 era: potential trends, challenges, and..., Türközer [/bib_ref] [bib_ref] Psychiatry and COVID-19, Öngür [/bib_ref]. Yet, it also provides a rare opportunity for researchers and clinicians to draw from-and contribute to-the growing literature on remote digital technologies in psychiatry. Digital technology promoting mental health research and practice, or e-mental health, has become prevalent worldwide and can improve the implementation of evidencebased practice [bib_ref] E-mental health: promising advancements in policy, research, and practice, Lal [/bib_ref] [bib_ref] WPA position statement on e-mental health, Wise [/bib_ref]. Most individuals with schizophrenia [bib_ref] Digital technology use among individuals with schizophrenia: results of an online survey, Gay [/bib_ref] and first-episode psychosis [bib_ref] Understanding access and use of technology among youth with first-episode psychosis to..., Abdel-Baki [/bib_ref] have access to a computer, smartphone, or tablet and growing research supports the use, acceptability, feasibility, and efficacy of digital technologies in psychiatry [bib_ref] Digital interventions for screening and treating common mental disorders or symptoms of..., Sin [/bib_ref] [bib_ref] Preferences of young adults with first-episode psychosis for receiving specialized mental health..., Lal [/bib_ref] [bib_ref] Digital technology for management of severe mental disorders in low-income and middle-income..., Merchant [/bib_ref] [bib_ref] Mobile apps for mental health issues: meta-review of metaanalyses, Lecomte [/bib_ref]. Digital cognitive assessments are also being increasingly developed for these devices, with recent reviews suggesting they are feasible and reliable measures of cognition [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] [bib_ref] Assessing cognition outside of the clinic: smartphones and sensors for cognitive assessment..., Hays [/bib_ref] [bib_ref] Verbal memory measurement towards digital perspectives in first-episode psychosis: a review. Schizophr, Kilciksiz [/bib_ref]. Remote cognitive assessments provide many opportunities to advance research and treatment in severe mental illness, particularly schizophrenia-spectrum disorders. As they are typically digital measures, remote assessments can benefit from advances in the field of computerized neuropsychological assessment (e.g., ref. [bib_ref] Computerized neuropsychological assessment devices: joint position paper of the American Academy of..., Bauer [/bib_ref] as evidenced more broadly by the InterOrganizational Practice Committee guidelines for teleneuropsychology [bib_ref] InterOrganizational practice committee recommendations/ guidance for teleneuropsychology (TeleNP) in response to the..., Bilder [/bib_ref]. Remote assessments also offer the same advantages as computerized measures, including increased precision, standardized testing, and automated scoring [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] [bib_ref] Applications of computer-based neuropsychological assessment, Schatz [/bib_ref] [bib_ref] The sensitivity and psychometric properties of a brief computer-based cognitive screening battery..., Langenecker [/bib_ref]. Moreover, they enable the recruitment of larger and more diverse samples (e.g., from rural and remote areas) and of individuals who might have practical (e.g., cost, transportation) or symptomatic (e.g., social avoidance, paranoia) issues that make in-person attendance difficult. Assessments using tablets and smartphones have added benefits in that they can more easily be completed remotely at any time and in any geographic location [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] [bib_ref] Mobile technology for cognitive assessment of older adults: a scoping review, Koo [/bib_ref] and can provide data on additional dynamic variables (e.g., environment data, sleep quality, mood, level of exercise, etc.) for a broader assessment of cognition [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref]. There is an urgent need to verify that remote cognitive assessments provide valid assessments of cognitive capacity and cognitive biases in severe mental illness. Although recent reviews support the use of digital cognitive assessments in psychiatric populations, delivery in remote settings is not yet common [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] [bib_ref] Assessing cognition outside of the clinic: smartphones and sensors for cognitive assessment..., Hays [/bib_ref] [bib_ref] Verbal memory measurement towards digital perspectives in first-episode psychosis: a review. Schizophr, Kilciksiz [/bib_ref]. Consequently, many researchers and clinicians are rapidly embarking on this path with little empirical evidence to provide guidance. The purpose of this scoping review is to provide an overview of the literature on remote cognitive assessment in severe mental illness. We focus on remote assessments in psychiatric illnesses rather than broad digital measures or remote measures in the general population given the great potential for remote assessments to drive research and treatment in this population [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] [bib_ref] Assessing cognition outside of the clinic: smartphones and sensors for cognitive assessment..., Hays [/bib_ref]. We opted for a scoping review as they are designed to address broad, overarching research questions within a systematic review framework [bib_ref] Systematic review or scoping review? Guidance for authors when choosing between a..., Munn [/bib_ref] [bib_ref] Conducting high quality scoping reviews-challenges and solutions, Khalil [/bib_ref]. Our main population of interest included individuals with severe mental illness (e.g., schizophrenia-spectrum disorders), though we did not exclude research involving other groups. Our objectives were to map the current literature, identify potential barriers and facilitators, and highlight knowledge gaps in remote cognitive assessment in severe mental illness. This review aims to provide insight into the currently available options for clinicians and researchers and encourage high-quality research on remote cognitive assessment in psychiatry during and beyond the COVID-19 pandemic. [fig_ref] Figure 1: PRISMA flow diagram of article selection and reasons for exclusion [/fig_ref] displays the PRISMA flowchart, combining the retrieved articles across the three literature searches. In the initial search, 24,516 references were identified, including one in press manuscript through a co-author (SG). After the removal of 1760 duplicates, titles and abstracts of 22,756 articles were randomly divided and screened by five reviewers. Of these, 57 articles were flagged as potentially relevant and full texts were screened. Upon full-text review, 32 additional articles were excluded due to not meeting one or more of the selection criteria. One additional article was identified through a reference list search. An updated search after 6 months yielded an additional 859 articles, five of which met inclusion criteria, with one additional article found through reference list search. A final updated search 3 months later yielded an additional 1124 articles (note: search updates were limited by year and overlapped with previous searches), two of which met inclusion criteria. Thus, 34 articles were included in the scoping review, including a narrative review of digital technology for remote cognitive assessment in psychiatry 26 , a commentary on remote digital cognitive assessment in schizophrenia [bib_ref] Towards remote digital phenotyping of cognition in schizophrenia, Guimond [/bib_ref] , and a systematic review on digital assessment of verbal memory in first-episode psychosis [bib_ref] Verbal memory measurement towards digital perspectives in first-episode psychosis: a review. Schizophr, Kilciksiz [/bib_ref]. These three nonexperimental articles are incorporated only into the facilitators, barriers, and future directions sections of the logic model and the remaining articles informed all sections of the model. # Results ## Selection of sources of evidence Inter-and Intra-rater reliability Inter-rater reliability (IRR) was high at start, midpoint and end of article selection and increased over time: IRR 1 = 0.95 (SE = 0.02, 95% CI = [0.92-0.98], p < 0.001, two-sided); IRR 2 = 0.97 (SE = 0.01, 95% CI = [0.94-1.00], p < 0.001, two-sided); IRR 3 = 0.98 (SE = 0.01, 95% CI = [0.96-1.00], p < 0.001, two-sided). Supplementary displays the distribution of disagreements (initial rating compared to consensus) per rater over the three IRRs. The number of articles (out of 100 at each timepoint) with conflicting ratings between two or more raters was low and decreased over time: IRR 1 = 10/ 100, IRR 2 = 6/100, IRR 3 = 4/100. The mean number of conflicts was also low and decreased (IRR 1 = 3.20, SD = 2.59; IRR 2 = 2.60, SD = 1.52; IRR 3 = 1.40, SD = 1.67). [fig_ref] Table 1: Primary characteristics for selected articles [/fig_ref] lists the 31 experimental articles selected for review (excluding the three review articles of the total selected 34 articles), along with primary characteristics (psychiatric diagnosis, sample size, remote platform, supervision, battery/measure assessed, and relevant cognitive domain). Full study characteristics are displayed in Supplementary Data, including sociodemographics (sample size, control group, age ranges, sex ratios, country, language), measure characteristics (study setting, researcher presence and title, license type, measure type, duration, alternate forms), psychometric properties, and sex-related findings. Selected articles were published between 2009 and 2021, though most (82.35%) were published within the past 5 years. ## Characteristics and results of sources of evidence ## Synthesis of results: logic model The final logic model is presented in [fig_ref] Figure 2: Final logic model of remote cognitive assessment measures in severe mental illness [/fig_ref]. The central panel includes 82 remote cognitive measures divided into 11 cognitive domains. The most assessed domains were speed of processing, working memory, reasoning, and executive function, whereas subjective cognition included only a single reviewed measure. For each measure, we illustrate which platform(s) were used (videoconference, web browser, tablet, and smartphone, in normal, bold, underline, and italic font, respectively) and whether the assessment was tested in a laboratory setting (white circle), remotely (black circle) or both (white and black circle). Briefly, two studies tested their measures using videoconferencing, 16 via web browser, two with a tablet, and nine with smartphones. Only one study [bib_ref] The validation of a new online cognitive assessment tool: the MyCognition Quotient, Domen [/bib_ref] reported their remote assessment could be performed on two platforms (i.e., tablet and web browser) through several used web-based measures that could likely be used on several platforms (e.g., web, smartphone, tablet). In total, six studies included remote measures that were completed in a laboratory setting, 23 were done remotely, and two used both settings. The upper circles of the logic model summarize reported reliability, sensitivity/specificity, construct validity, and criterion validity of the reviewed measures, detailed in Supplementary Data. For each cognitive domain, we report the number of times a given psychometric was evaluated over the total number of times it was measured across studies. Next to each total, we summarize the reported psychometric properties as either low (L), moderate (M), or high (H) and invite the reader to consult Supplementary Data for detailed findings. Reliability includes estimates of internal consistency, test-retest evaluations, and intraclass correlations. Sensitivity and specificity respectively refer to the ability of the reviewed measure to identify those with and without impairments. Construct validity includes correlations with comparison measures (e.g., pen-and-paper versions) and correlations between human and automated scoring. Criterion validity includes correlations between the reviewed measures and outcomes, such as sociodemographics, symptoms, and functioning. Construct validity was most frequently assessed irrespective of the cognitive domain, whereas reliability was assessed least frequently. Overall, we observe that, for measures in which psychometric properties are assessed, remote measures were generally as reliable, sensitive, and valid as traditional measures. One exception was social cognition, which showed poor discriminatory power in one study [bib_ref] Development and testing of a web-based battery to remotely assess cognitive health..., Biagianti [/bib_ref] and low to moderate correlations with traditional measures (see Supplementary Data). The lower panels of the logic model outline thematically defined barriers and facilitators to the development and implementation of remote cognitive assessment as well as proposed improvements and avenues for future research. For development, facilitators included incorporating standardized procedures, alternate measure versions, and using technology to mitigate potential barriers (e.g., preloading stimuli to limit internet connectivity issues). On the other hand, developmental barriers included confidentiality concerns, technology/system variability, imprecise measure adaptations, and the current lack of remote norms. For implementation, testing in a neutral setting, improving feasibility (reminders, user-friendly technology), and wider access to individuals living in rural regions have been identified as facilitators. Inversely, low participant engagement, symptom severity, limited digital literacy, poor technology accessibility, and potential access to outside help (e.g., through family members or the internet) have been identified as barriers. As for proposed improvements and future directions, the authors of reviewed studies highlighted the need for further psychometric validation, development of remote norms, and strategies to ensure digital security. There were also proposed improvements pertaining to the promotion of open-source options, optimization of collected data (detailed cognitive performance data and additional contextual variables, such as sleep and physical activity), and verification of diagnostic and cultural generalizability. ## Sex and gender considerations Given the well-documented sex differences in cognition and their relevance to psychiatric illness 37,38 , we sought to examine the role of sex and gender on remote assessment of cognitive capacity and cognitive biases. Approximately one-quarter of experimental studies (n = 9) reported on differences based on sex assigned at birth (male, female) and none on gender identity (e.g., non-binary, trans-, cis-, genderfluid). Sex and gender were often used interchangeably presumably in reference to sex assigned at birth. One study reported matching participants based on sex and used sex-corrected pen-and-paper norms [bib_ref] Validation of the tablet-administered brief assessment of cognition (BAC App), Atkins [/bib_ref] , one did not report explicit sex ratios [bib_ref] Originally adapted mobile application used for neuropsychiatric patients, Pop-Jordanova [/bib_ref] , and one included females only [bib_ref] Posttraumatic stress disorder symptoms and cognitive function in a large cohort of..., Sumner [/bib_ref]. Those that reported on sex differences found that females displayed higher cognitive biases [bib_ref] Mediating role of cognitive biases, resilience and depressive symptoms in the relationship..., Metel [/bib_ref] and lower performance on working memory 43 . Two articles described nonspecific sex differences [bib_ref] Development and testing of a web-based battery to remotely assess cognitive health..., Biagianti [/bib_ref] [bib_ref] Dysfunctional beliefs in patients with obsessivecompulsive disorder and depression as assessed with..., Miegel [/bib_ref] , and three found no sex-related performance [bib_ref] Originally adapted mobile application used for neuropsychiatric patients, Pop-Jordanova [/bib_ref] [bib_ref] Prolonged rather than hasty decision-making in schizophrenia using the box task. Must..., Moritz [/bib_ref] or attrition 46 differences (see . # Discussion The present study provides a scoping review of the literature on remote assessment of cognitive capacity and cognitive biases in severe mental illness to map current knowledge and inform clinicians and researchers on best practices. In total, more than 26,000 articles were retrieved and 34 met our inclusion criteria. Identified measures generally showed acceptable psychometric properties, though these were assessed in less than half of reviewed studies. Facilitators and barriers to the development and implementation of remote cognitive assessment measures, as well as future research directions proposed by identified studies, provide clear considerations for future research and practice. This work brings together the current library of remote cognitive measures in psychiatry that researchers and clinicians may consult based on their needs, including cognitive domain, remote platform, and level of supervision required. Below we provide general recommendations and considerations to foster remote cognitive assessment in psychiatry. Our scoping review did not identify a "gold-standard" remote battery for a comprehensive assessment of cognition in psychiatric populations. Moreover, there is currently no single cognitive battery, remote or otherwise, assessing both cognitive capacity and cognitive biases to provide an overall measure of cognitive health in severe mental illness. For cognitive capacity, the two most frequently used computerized cognitive batteries in psychiatric populations (CANTAB and CogState) [bib_ref] Systematic review of appropriate cognitive assessment instruments used in clinical trials of..., Bakkour [/bib_ref] did not emerge strongly in our review, suggesting they have not yet been adopted extensively in remote settings despite their potential for remote administration. Only one study [bib_ref] Posttraumatic stress disorder symptoms and cognitive function in a large cohort of..., Sumner [/bib_ref] used the CogState Brief Battery in a remote setting in a very large sample of nurses with elevated PTSD symptoms, though the generalizability of the results to other psychiatric samples remains in question. CANTAB was only used in a single study as an in-lab comparison measure [bib_ref] The validation of a new online cognitive assessment tool: the MyCognition Quotient, Domen [/bib_ref]. Moreover, social cognition measures were restricted to emotion recognition tasks and tests of other domains of social cognition (e.g., theory of mind) are currently lacking. Notable comprehensive remote batteries that reported acceptable psychometric properties included the Brief Assessment of Cognition 39 , My Cognition Quotient 35 , Online Neurocognitive Assessments 36 , and Screen for Cognitive Assessment in Psychiatry [bib_ref] Cyber-Neuropsychology: application of new technologies in neuropsychological evaluation, Bernardo-Ramos [/bib_ref]. Some individual tasks also showed valid, sensitive, and/or reliable remote administration, particularly the Jewel Trail Making Task from the mindLAMP smartphone application, used in three studies [bib_ref] Validation of an ecological momentary assessment to measure processing speed and executive..., Schvetz [/bib_ref] [bib_ref] Assessing the potential of longitudinal smartphone based cognitive assessment in schizophrenia: a..., Liu [/bib_ref] [bib_ref] Deriving symptom networks from digital phenotyping data in serious mental illness, Hays [/bib_ref]. Cognitive biases were primarily assessed using scales rather than tasks, which are more amenable to remote administration via online survey platforms. Importantly, most cognitive bias scales and all cognitive bias tasks identified were designed to address individual biases, such as jumping to conclusions [bib_ref] Prolonged rather than hasty decision-making in schizophrenia using the box task. Must..., Moritz [/bib_ref] [bib_ref] Negative affect and a fluctuating jumping to conclusions bias predict subsequent paranoia..., Ludtke [/bib_ref] [bib_ref] The benefits of doubt: cognitive bias correction reduces hasty decision-making in schizophrenia, Moritz [/bib_ref] [bib_ref] Metacognition-augmented cognitive remediation training reduces jumping to conclusions and overconfidence but not..., Moritz [/bib_ref]. The most general measure of cognitive biases identified was the Davos Assessment of Cognitive Biases Scale 55 , though it does not measure all biases reported in psychiatric disorders. Surprisingly, the well-known Cognitive Biases Questionnaire for Psychosis 56 did not emerge in our review, suggesting it has yet to be used in remote settings with severe mental illness. Given the importance of cognitive biases in understanding and treating the symptoms of severe mental illness 7 , the development of a validated remote cognitive bias battery to complement the numerous batteries that exist to assess cognitive capacity is recommended. Fundamentally, the question of which measure(s) to use depends on the cognitive domain(s) of interest and other pragmatic considerations (platform, duration, cost, etc). Comprehensive batteries would likely be most convenient for clinicians and for researchers interested in general measures of cognition across various domains. However, most of the available comprehensive cognitive batteries are proprietary (Supplementary Data) and thus incur significant costs and less flexibility for the user. Several open-source measures were available through online platforms, such as Inquisit Web or researcher-developed applications. There exist other promising experiment-sharing platforms (e.g., Pavlovia, Expyriment, CognitionLab), though, to our knowledge, these have yet to be tested remotely with psychiatric samples. Generally, these platforms require "picking and choosing" and/or developing cognitive measures and thus necessitate greater reflection on the objectives and cognitive measures of interest. True open-source alternatives, in which the task's source code is fully accessible are also available for some measures, or reportedly available from the authors. These initiatives would likely be of greater interest to cognitive scientists. While this review illustrates that remote cognitive assessment is feasible with psychiatric populations, most studies strongly recommended further validation of existing remote measures, development of additional measures, and remote norms. Remote norms were not reported in the identified studies, despite the potential for remote studies to collect data from large and diverse samples and the growing number of computerized batteries with normative data (e.g., refs. [bib_ref] Is the Web as good as the lab? Comparable performance from Web..., Germine [/bib_ref] [bib_ref] Comparison of cognitive performance on the Cogstate brief battery when taken in-clinic,..., Cromer [/bib_ref] [bib_ref] Comparing web-based and lab-based cognitive assessment using the Cambridge Neuropsychological Test Automated..., Backx [/bib_ref] [bib_ref] Development and validation of a World-Wide-Web-based neurocognitive assessment battery: WebNeuro, Silverstein [/bib_ref]. Only one selected study assessed whether in-lab computerized scores were comparable to pen-andpaper norms, finding that modifications were necessary for some subtests of the Brief Assessment of Cognition 39 . Thus, normative data derived from in-person assessments might not be applicable to computerized or remote versions of all cognitive tests. The development of remote norms would greatly facilitate remote cognitive assessment and allow for improved comparisons between studies. However, this poses several challenges. Notably, comparable in-person normative data are not available for all tests, particularly for measures of cognitive biases. In addition, the nature of remote assessment occurring outside the laboratory naturally reduces researchers' control over environmental confounds that could affect test performance. Future development of remote normative data and guidelines for such norms should address these potential issues. Additional quality considerations should be made during both the development and implementation of a new cognitive task or study. In terms of development, identified studies strongly encouraged using standardized and automated procedures, including instructions and scoring, to reduce variability and human error. Moreover, eliminating the need for a synchronous internet connection (e.g., preloading cognitive stimuli and allowing test results to be uploaded asynchronously) can mitigate potential issues with internet connectivity. Adaptation of certain pen-and-paper measures to remote computerized software also presents a major challenge to validity and feasibility, particularly for those measures that involve writing or motor skills, and penand-paper norms may be inaccurate in these cases. The choice of remote platform (web, tablet, smartphone, videoconference) or multi-platform options should also be carefully evaluated, as platforms vary in terms of functionality (e.g., touch screen ability) and other parameters (e.g., screen size, computational power) that can affect performance. It is also imperative to ensure that collected data corresponds to high ethical standards in terms of security and privacy, including transparency, confidentiality, data safeguarding, and avoiding superfluous data collection [bib_ref] Navigating the ethics of remote research data collection, Gelinas [/bib_ref] [bib_ref] Remote data collection for public health research in a COVID-19 era: ethical..., Hensen [/bib_ref]. Finally, when implementing cognitive assessments in remote settings, participants' digital competence, symptom severity, and potential environmental distractors should be considered, all of which can affect performance over and above cognitive impairments. Reminder notifications, standardized instructions, practice, and remote monitoring may limit these potential issues. Future remote studies should prioritize larger samples, standardization of instructions and environment, where possible, broader data collection (e.g., environmental data, sleep quality, mood, level of exercise, etc.) and wider recruitment (e.g., remote and rural areas) to allow for the development of norms and to assess potential sociodemographic factors (sex, gender, race, education, etc.) and diagnostic and cultural generalizability. Development and validation of additional remote measures of both cognitive capacity and cognitive biases would also bring us closer to developing an overall battery of cognitive health for those with psychiatric disorders. Quality remote cognitive assessments have strong implications for remote cognitive interventions in psychiatry. Effective cognitive interventions are available for both cognitive capacity (e.g., cognitive remediation therapy) [bib_ref] Action-based cognitive remediation for individuals with serious mental illnesses: effects of realworld..., Bowie [/bib_ref] [bib_ref] Combined cognitive remediation and functional skills training for schizophrenia: effects on cognition,..., Bowie [/bib_ref] [bib_ref] Cognitive remediation for negative symptoms of schizophrenia: a network meta-analysis, Cella [/bib_ref] [bib_ref] A meta-analysis of cognitive remediation for schizophrenia: methodology and effect sizes, Wykes [/bib_ref] and cognitive biases (e.g., metacognitive training, cognitive behavioral therapy for psychosis) [bib_ref] Efficacy of psychological interventions targeting cognitive biases in schizophrenia: a systematic review..., Sauvé [/bib_ref] [bib_ref] Acceptance and efficacy of metacognitive training (MCT) on positive symptoms and delusions..., Eichner [/bib_ref]. In a complimentary review and meta-analysis on the efficacy of virtual evidence-based psychosocial interventions for schizophrenia-spectrum disorders 69 , 11 studies met inclusion criteria for virtually-delivered cognitive remediation. Six of these were included in a meta-analysis showing moderate effects on neurocognition (Hedges g = 0.35) and functioning (g = 0.33), similar to in-person interventions [bib_ref] A meta-analysis of cognitive remediation for schizophrenia: methodology and effect sizes, Wykes [/bib_ref]. These initial results on efficacy are promising for virtual adaptations of existing interventions and encourage the development of new programs specifically designed for virtual delivery. For example, patient-tailored remote interventions following a preliminary remote cognitive assessment would integrate personalized treatment and broad accessibility. The current study presents several strengths. First, it is a broad scoping review of remote measures of both cognitive capacity and cognitive biases in severe mental illness designed to address an urgent need given the COVID-19 pandemic. Second, it involves rigorous methodological procedures including randomization, repeated inter-rater reliability, extensive quality control, and iterative data synthesis. Third, the search was updated after six and nine months given the rapidly evolving literature in this domain. Finally, data extraction was comprehensive and included several characteristics (e.g., diagnosis, setting, researcher presence, platform, duration, alternate forms, licensing, cognitive domain, psychometric properties) that will assist researchers and clinicians in their choice of remote measures. A potential limitation of this study is that the search strategy, which was focused on severe mental illness, may not have captured all articles assessing remote cognition in other psychiatric disorders, though several were identified, and reference lists were also checked. Additionally, we did not calculate quality scores for included studies. Contrary to systematic literature reviews, a critical appraisal of sources of evidence is not generally indicated for scoping reviews, which are meant to be broadly inclusive of the literature [bib_ref] Updated methodological guidance for the conduct of scoping reviews, Peters [/bib_ref]. Third, despite our best efforts, our review may have missed some findings from unpublished studies and ongoing investigations. This is particularly relevant given the present surge in remote research due to the COVID-19 pandemic and is illustrated by the eight additional sources of evidence identified in our updated searches. There are also many additional remote cognitive measures and batteries that were identified during the review process, but these had not yet been tested in populations with severe mental illness and were outside the scope of this review. Lastly, our domain classifications may not accurately represent all cognitive function(s) assessed by a given measure. However, this classification was developed using an iterative process until consensus was reached by the three lead authors and was reviewed and approved by the remaining authors, all of whom are experienced in the field. At present, researchers and clinicians in psychiatry can choose from a vast selection of remote cognitive measures assessing many cognitive domains through various remote platforms. However, there is an urgent need for more rigorous validation of these measures and for a stronger consideration of influential factors, such as sex and gender differences and cultural diversity. Remote cognitive assessment is necessary given the current climate but also has the potential to propel the field of cognitive psychiatry forward. In conclusion, this review provides clinicians and researchers with a comprehensive list of remote cognitive assessment measures as well as insight into methodological and practical considerations that may serve as a first step in the development of guidelines for remote cognitive assessment in severe mental illness. # Methods ## Protocol and registration The review protocol was preregistered on the Open Science Framework: https://osf.io/cbzq8 (Registration 10.17605/OSF.IO/ CBZQ8) and followed the PRISMA extension for scoping reviews 71 (see for PRISMA checklist) and the Joanna Briggs Institute guidance on conducting systematic scoping reviews [bib_ref] Conducting high quality scoping reviews-challenges and solutions, Khalil [/bib_ref] [bib_ref] Updated methodological guidance for the conduct of scoping reviews, Peters [/bib_ref] [bib_ref] Chapter 11: Scoping Reviews, Peters [/bib_ref]. Search strategy and selection criteria A comprehensive literature search was conducted on May 11, 2020 and updated on November 11, 2020, and February 4, 2021 using OVID (MEDLINE, PsycInfo, and EMBASE) and EBSCO (CINAHL) databases. The following keywords were used: (schizophreni* OR psychosis OR psychoses OR psychotic* OR severe mental illness) AND (cogniti* OR neuropsych* OR bias* OR reason*) AND (remote* OR online* OR mobile* OR digital*) AND (assessment OR evaluat* OR test* OR measure*). The search was limited to articles in either English or French from any publication year. Evidence sources included peer-reviewed research articles, reviews, and letters to the editor, excluding books and conference abstracts. Repositories of tests and measures were searched (PsycTESTS, Health and Psychosocial Instruments, Mental Measurements Yearbook), experts were contacted for unpublished findings, and reference lists of selected articles were examined for additional studies. ## Article screening Retrieved articles were combined in Endnote software, and in a first pass, duplicates were excluded automatically by comparing Author, Year, Title, and Journal fields. Duplicates based on all possible other combinations of these fields were produced and checked manually. The remaining articles were randomized for initial screening based on title and abstract. Due to the urgent nature of this review, five raters were assigned to screen the remaining de-duplicated articles, with each rater screening approximately one-fifth of the total number of de-duplicated articles. Raters assigned each article one of three possible ratings: include, questionable, exclude. To determine whether questionable articles should be included or excluded, full texts were reviewed according to the study inclusion criteria and a consensus was reached by the research team. Article screening was based on the following eligibility criteria: (a) peer-reviewed; (b) included individuals with a diagnosis involving severe mental illness (e.g., schizophrenia-spectrum disorders); and (c) reported on the remote assessment of cognitive capacity and/or cognitive biases. During article selection, we recognized that several articles included a broad range of diagnostic groups (e.g., anxiety, depression, OCD) and we included these conditions to maintain a broader scope. In addition, many articles assessed remote cognitive tasks in a laboratory setting (e.g., comparison with a standard pen-and-paper battery). In order to include these articles, which were not technically remote, while not including all articles reporting on computerized cognitive assessment in psychiatry, we included these on a case-by-case basis, and the inclusion of articles determined via consensus. Selected articles were then retrieved for full-text screening and data extraction of included articles. Given that articles were screened by different raters, rather than by all raters, we assessed inter-rater reliability (IRR) by having all raters assign ratings to three samples of 100 articles at the start, midpoint, and end of article selection, as in previous research [bib_ref] Interrater reliability in systematic review methodology: exploring variation in coder decision-making. Sociol, Belur [/bib_ref]. IRR was calculated using Gwet's AC 1 statistic 74 via the R AgreeStat package to account for the kappa paradox, in which unusually low agreement statistics are produced when there is a skewed distribution of ratings (e.g., many excluded articles) [bib_ref] High agreement but low kappa: I. The problems of two paradoxes, Feinstein [/bib_ref] [bib_ref] A comparison of Cohen's Kappa and Gwet's AC1 when calculating inter-rater reliability..., Wongpakaran [/bib_ref] [bib_ref] High agreement and high prevalence: the paradox of Cohen's Kappa, Zec [/bib_ref] [fig_ref] Table 1: Primary characteristics for selected articles [/fig_ref] for a demonstration with the current data). Following each IRR timepoint, raters produced a consensus for any inconsistent ratings. Intra-rater reliability was also assessed across IRR timepoints 73 by comparing each rater's accuracy relative to consensus. ## Data extraction Data extraction was performed on selected articles according to a pre-developed form, which was tested and fine-tuned with one exemplar article by the lead author. Articles were randomized for data extraction across three independent raters. Data extraction was quality controlled by authors K.M.L., G.S., and D.R.-C. by randomly selecting six articles (10% of articles originally extracted) and re-extracting the data. Data extraction included the following predetermined variables: bibliographic data (authors, year, title, abstract), study characteristics (aims, design, country, setting, researcher presence/title, sample size, psychiatric diagnosis, mean age, age range, sex/gender ratio), description of remote assessment methods (remote/comparison measure(s), battery, remote platform, developer, language, duration, alternate forms, availability of norms), main findings, sex/gender findings, psychometric properties (reliability, sensitivity/specificity, construct validity, criterion validity), facilitators, barriers, and future directions. # Synthesis of results Data were synthesized and illustrated using the logic model methodology, following the W. K. Kellogg Foundation guidelinesand previous research [bib_ref] Using logic model methods in systematic review synthesis: describing complex pathways in..., Baxter [/bib_ref] [bib_ref] How do recovery-oriented interventions contribute to personal mental health recovery? A systematic..., Winsper [/bib_ref]. This flexible method uses an iterative approach to identify and illustrate thematic categories and the putative underlying links to portray complex relationships [bib_ref] Creating and using logic models, Conrad [/bib_ref]. In this study, the logic model was used to classify cognitive measures into domains (speed of processing, attention and vigilance, working memory, verbal learning and memory, visual learning and memory, reasoning and executive function, social cognition, verbal fluency, cognitive bias, subjective cognition, and IQ), expanded from the MATRICS 83 classification. The logic model also outlines psychometric properties, facilitators, barriers, and future directions identified. ## Logic model development Cognitive measures were categorized into cognitive domains, which were inspired by the MATRICS 83 classification: speed of processing, attention and vigilance, working memory, verbal learning and memory, visual learning and memory, reasoning and problem solving, and social cognition. We added verbal fluency, cognitive bias, subjective cognition, and IQ domains, to account for identified measures which did not fit within the MATRICS domains. We initially selected the MATRICS classification as it provides a well-known framework for cognitive impairment in schizophrenia, which was our primary population of interest and the group assessed in most studies. In addition, the MATRICS domain of reasoning and problem solving was relabeled as "reasoning and executive function" in order to include additional measures of executive functioning (e.g., inhibitory control) without creating a separate domain. Notably, several measures tap into additional domains reported in the literature (e.g., visuomotor processing) or recruit additional cognitive processes that fall into other identified domains (e.g., speed of processing measures also require attention). In the current review, a given measure's primary cognitive domain is reported and was determined through consensus. Differences between draft and final logic model Prior to data extraction, we developed a draft logic model [fig_ref] Figure 1: PRISMA flow diagram of article selection and reasons for exclusion [/fig_ref]. The final logic model [fig_ref] Figure 2: Final logic model of remote cognitive assessment measures in severe mental illness [/fig_ref] was developed through an iterative process by the three lead authors and was reviewed and approved by the remaining authors. Differences between the draft and final logic models are outlined below. In both models, the identified remote cognitive measures, relevant cognitive domains, and procedural characteristics are displayed in the middle panel. Psychometric properties are located in the upper circles and facilitators, barriers, and future directions in the lower sections. The draft logic model categorized the identified remote cognitive assessment measures categorized by MATRICS cognitive domain (speed of processing, attention/vigilance, working memory, verbal memory and learning, visual memory and learning, reasoning and problem solving, social cognition) [bib_ref] The MATRICS Consensus Cognitive Battery, part 1: test selection, reliability, and validity, Nuechterlein [/bib_ref] with the addition of a cognitive bias domain. The draft model also outlined utilized procedures (setting, platform, researcher presence/title, duration, material, cost), psychometric properties (reliability, sensitivity/specificity, concurrent validity, predictive validity), facilitators, barriers, improvements/future research, and recommendations. During data extraction, we decided to report on the license type (proprietary, open-source) of a given measure/battery, rather than cost, as this was not readily available. To simplify the presentation, the final logic model reports only the platform and location of testing with the other procedures relegated to Supplementary Data. We also renamed concurrent and predictive validity to construct and criterion validity, respectively, to emphasize the wider breadth of psychometric properties that were available. Facilitators, barriers, improvements/future research remained unchanged from the draft to the final logic model. ## Data availability Data generated from this study are available via the Open Science Framework (OSF; https://osf.io/wh6vt/) with the identifier https://doi.org/10.17605/OSF.IO/WH6VT 84 . ## Code availability Inter-rater reliability was calculated with the AgreeStat package in R, available at: https://agreestat.com/software/r/new/agree.coeff3.raw.r. [fig] Figure 1: PRISMA flow diagram of article selection and reasons for exclusion. Numbers from the three searches (May 11, 2020, November 11, 2020, and February 4, 2021) are combined in this figure but described separately in the main text. N number of articles. [/fig] [fig] Figure 2: Final logic model of remote cognitive assessment measures in severe mental illness. Middle panel lists remote cognitive measures, tested platform (tablet, web, videoconference, and/or smartphone), and study type (remote, in-lab, or both) per cognitive domain. Upper circles represent the number of measures per cognitive domain in which psychometric properties (reliability, sensitivity/specificity, construct/ criterion validity) were assessed over the number of measures assessing that domain. Adjacent letters summarize psychometric properties (low, L, moderate, M, high, H) detailed in Supplementary Data. Lower panels summarize facilitators, barriers, and avenues for future research, which are meant to guide future remote cognitive assessment. K.M. Lavigne et al. [/fig] [table] Table 1: Primary characteristics for selected articles. [/table]