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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]
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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]
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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]
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